Glucokinase (GCK) iRNA compositions and methods of use thereof

ABSTRACT

The invention relates to double stranded ribonucleic acid (dsRNA) compositions targeting a glucokinase (GCK) gene, as well as methods of inhibiting expression of a glucokinase (GCK) gene, and methods of treating subjects having a glycogen storage disease (GSD), e.g., type Ia GSD.

RELATED APPLICATIONS

This application is a 35 § U.S.C. 111(a) continuation application which claims the benefit of priority to PCT/US2016/038616, filed on Jun. 22, 2016, which claims the benefit of priority of U.S. Provisional Patent Application No. 62/183,413, filed on Jun. 23, 2015, U.S. Provisional Patent Application No. 62/200,207, filed on Aug. 3, 2015, and U.S. Provisional Patent Application No. 62/260,876, filed on Nov. 30, 2015. The entire contents of each of the foregoing applications are hereby incorporated herein by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Dec. 13, 2017, is named 121301-03704_SL.txt and is 523,732 bytes in size.

BACKGROUND OF THE INVENTION

Hypoglycemia is a biochemical symptom indicating the presence of an underlying disease or disorder. Since glucose is the fundamental energy currency of the cell, diseases and disorders that affect its availability or use can cause hypoglycemia.

The body normally defends against hypoglycemia by decreasing insulin secretion and increasing glucagon, epinephrine, growth hormone, and cortisol secretion. These hormonal changes combine to increase hepatic glucose production, increase alternative fuel availability, and decrease glucose use. The increase in hepatic glucose production is initially caused by the breakdown of liver glycogen stores resulting from lower insulin levels and increased glucagon levels. When glycogen stores become depleted and protein breakdown increases because of increased cortisol levels, hepatic gluconeogenesis replaces glycogenolysis as the primary source of glucose production. Decreased use of peripheral glucose occurs initially because of a fall in insulin levels and later because of increases in epinephrine, cortisol, and growth hormone levels.

All of these events increase lipolysis and plasma free fatty acid levels, which are then available as an alternative fuel source and act to competitively inhibit glucose use. Plasma free fatty acids also stimulate glucose production. Hypoglycemia occurs when one or more of these counterregulatory mechanisms fail because of, for example, the overuse of glucose (as in hyperinsulinism) or the underproduction of glucose (as in a glycogen-storage diseases).

GSD is an inherited genetic disorder due to an absence or deficiency of one of the enzymes responsible for making or breaking down glycogen in the body. This enzyme deficiency causes either abnormal tissue concentrations of glycogen or incorrectly or abnormally formed glycogen and patients with glycogen storage diseases (GSD) may also have low blood glucose levels. GSD occurs in about one of 50,000 to 100,000 births. Some patients might die before diagnosis, while severe infantile forms and some milder forms might go unrecognized Symptoms of GSD vary based on the enzyme that is missing and usually result from the buildup of glycogen or from the inability to produce glucose when needed. Because GSD occurs mainly in muscles and the liver, those areas show the most symptoms, such as, poor growth, muscle cramps, low blood sugar, enlarged liver, swollen belly, and abnormal blood chemistry.

Current treatments for glycogen storage diseases have been very limited and mainly focused on correcting hypoglycemia and other metabolic disturbances through dietary control, such as, by using a modified form of cornstarch, or a high-protein diet.

Accordingly, there is a need in the art for alternative treatments for subjects having a glycogen storage disease (GSD), e.g., type Ia GSD, which are independent of the underlying molecular defects.

SUMMARY OF THE INVENTION

The present invention provides iRNA compositions which effect the RNA-induced silencing complex (RISC)-mediated cleavage of RNA transcripts of a glucokinase (GCK) gene. The GCK gene may be within a cell, e.g., a cell within a subject, such as a human. The present invention also provides methods of using the iRNA compositions of the invention for inhibiting the expression of a GCK gene and/or for treating a subject who would benefit from inhibiting or reducing the expression of a GCK gene, e.g., a subject suffering or prone to suffering from a glycogen storage disease (GSD), or one or more signs or symptoms of GSD, such as hypoglycemia, lactic acidosis, hyperuricemia, hyperlipidemia, hepatomegaly, kidney disease as a result if glycogen accumulation, hunger, jitteriness, lethargy, apnea, seizures, diaphoresis, confusion, headaches, dizziness, unusual mood or behavior changes, loss of consciousness, coma, muscle cramps, bleeding diathesis, short stature, osteoporosis, delayed puberty, gout, renal disease, systemic hypertension, pulmonary hypertension, hepatic adenomas, pancreatitis, anemia, vitamin D deficiency, polycystic ovaries, irregular menstrual cycles, menorrhagia, and/or eruptive xanthomata.

GCK in the liver of a subject is the rate limiting enzyme for both glucose uptake and glycogen synthesis. Furthermore, the liver is a major site for regulation of whole body glucose metabolism. Therefore, by inhibiting expression of a glucokinase gene, e.g., a glucokinase gene in the liver of a subject, with a double stranded RNAi agent of the invention, e.g., an RNAi agent that contains and/or is coupled to a ligand, e.g., GalNAc3, that directs the RNAi agent to a site of interest, e.g., the liver, hepatic glucose uptake and storage will decrease, thus, inhibiting hypoglycemia, e.g., fasting hypoglycemia, in subjects suffering or prone to suffering from a glycogen storage disease (GSD), e.g., type Ia GSD, independent of the underlying cause. Furthermore, by inhibiting expression of a glucokinase gene, e.g., a glucokinase gene in the liver of a subject, with a double stranded RNAi agent of the invention, insulin resistance in the liver is selectively induced without altering insulin clearance in order to maintain safe glucose levels in subjects suffering or prone to suffering from a glycogen storage disease (GSD), e.g., type Ia GSD. In addition, by inhibiting expression of a glucokinase gene, e.g., a glucokinase gene in the liver of a subject having a glycogen storage disease (GSD), e.g., type Ia GSD, with a double stranded RNAi agent of the invention, hepatic glucose uptake and storage will decrease, thus, inhibiting hypoglycemia, e.g., fasting hypoglycemia, inhibit hepatic glycogen storage to inhibit hepatomegaly, reduce lipid abnormalities, lactic acidosis and/or hyperuricemia.

Accordingly, in one aspect, the present invention provides double stranded ribonucleic acids (RNAi) agents for inhibiting expression of a glucokinase (GCK) gene. The double stranded RNAi agents comprise a sense strand and an antisense strand, wherein the sense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from the nucleotide sequence of SEQ ID NO:1 and the antisense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from the nucleotide sequence of SEQ ID NO:15.

In another aspect, the invention provides double stranded ribonucleic acids (RNAi) agents for inhibiting expression of glucokinase (GCK), wherein the double stranded RNAi agents comprises a sense strand and an antisense strand, the antisense strand comprising a region of complementarity which comprises at least 15 contiguous nucleotides differing no more than 3 nucleotides from any one of the antisense sequences listed in any one of Tables 2, 3, 6, and 7.

In certain embodiments, the double stranded RNAi agent comprises at least one modified nucleotide. In one embodiment, the modified nucleotide comprises a 2′-O-methyl modified nucleotide. In another embodiment, the modified nucleotide comprises a 2′-fluoro modified nucleotide. In one embodiment, the modified nucleotide comprises a 3′-terminal deoxy-thymine (dT) nucleotide. In another embodiment, the modified nucleotide comprises a short sequence of deoxy-thymine (dT) nucleotides.

In certain embodiments, the dsRNA comprises no more than 4 (i.e., 4, 3, 2, 1, or 0) unmodified nucleotides in the sense strand. In certain embodiments, the dsRNA comprises no more than 4 (i.e., 4, 3, 2, 1, or 0) unmodified nucleotides in the antisense strand. In certain embodiments, the double stranded RNAi agent comprises no more than 4 (i.e., 4, 3, 2, 1, or 0) unmodified nucleotides in both the sense strand and the antisense strand. In certain embodiments, the double stranded RNAi agent comprises all modified nucleotides in the sense strand. In certain embodiments, the dsRNA comprises all modified nucleotides in the antisense strand. In certain embodiments, the double stranded RNAi agent comprises all modified nucleotides in both the sense strand and the antisense strand.

In one aspect, the present invention provides double stranded ribonucleic acid (RNAi) agents for inhibiting expression of glucokinase (GCK), wherein the double stranded RNAi agent comprises a sense strand and an antisense strand forming a double stranded region, wherein the sense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from the nucleotide sequence of SEQ ID NO:1 and the antisense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from the nucleotide sequence of SEQ ID NO:15, wherein substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand are modified nucleotides, and wherein the sense strand is conjugated to a ligand attached at the 3′-terminus.

In certain embodiments, at least one of the modified nucleotides is selected from the group consisting of a 3′-terminal deoxy-thymine (dT) nucleotide, a 2′-O-methyl modified nucleotide, a 2′-fluoro modified nucleotide, a 2′-deoxy-modified nucleotide, a locked nucleotide, an unlocked nucleotide, a conformationally restricted nucleotide, a constrained ethyl nucleotide, an abasic nucleotide, a 2′-amino-modified nucleotide, a 2′-0-allyl-modified nucleotide, 2′-C-alkyl-modified nucleotide, 2′-hydroxyl-modified nucleotide, a 2′-methoxyethyl modified nucleotide, a 2′-O-alkyl-modified nucleotide, a morpholino nucleotide, a phosphoramidate, a non-natural base comprising nucleotide, a tetrahydropyran modified nucleotide, a 1,5-anhydrohexitol modified nucleotide, a cyclohexenyl modified nucleotide, a nucleotide comprising a 5′-phosphorothioate group, a nucleotide comprising a 5′-methylphosphonate group, a nucleotide comprising a 5′ phosphate or 5′ phosphate mimic, a nucleotide comprising vinyl phosphate, a nucleotide comprising adenosine-glycol nucleic acid (GNA), a nucleotide comprising thymidine-glycol nucleic acid (GNA) S-Isomer, a nucleotide comprising 2-hydroxymethyl-tetrahydrofurane-5-phosphate, a nucleotide comprising 2′-deoxythymidine-3′phosphate, a nucleotide comprising 2′-deoxyguanosine-3′-phosphate, and a terminal nucleotide linked to a cholesteryl derivative or a dodecanoic acid bisdecylamide group.

In certain embodiments, the modified nucleotides is/are independently selected from the group consisting of a 2′-O-methyl modified nucleotide, a nucleotide comprising a 5′-phosphorothioate group, a 3′-terminal deoxy-thymine (dT) nucleotide, and a terminal nucleotide linked to a cholesteryl derivative or a dodecanoic acid bisdecylamide group. In certain embodiments, the modified nucleotide is selected from the group consisting of a 2′-deoxy-2′-fluoro modified nucleotide, a 2′-deoxy-modified nucleotide, a 3′-terminal deoxy-thymine (dT) nucleotide, a locked nucleotide, an abasic nucleotide, a 2′-amino-modified nucleotide, a 2′-alkyl-modified nucleotide, a morpholino nucleotide, a phosphoramidate, and a non-natural base comprising nucleotide.

In certain embodiments, the double stranded RNAi agent comprises a region of complementarity at least 17 nucleotides in length. In certain embodiments, the double stranded RNAi agent comprises a region of complementarity 19-23 nucleotides in length. In certain embodiments, the double stranded RNAi agent comprises a region of complementarity 19-21 nucleotides in length. In certain embodiments, the double stranded RNAi agent comprises a region of complementarity is 19 nucleotides in length. In certain embodiments, the double stranded RNAi agent comprises a region of complementarity is 21 nucleotides in length.

In certain embodiments, each strand of the double stranded RNAi agent is no more than 30 nucleotides in length. In certain embodiments, the double stranded RNAi agent is at least 15 nucleotides in length.

In some embodiments, at least one strand of the dsRNA agent comprises a 3′ overhang of at least 1 nucleotide, e.g., at least one strand comprises a 3′ overhang of at least 2 nucleotides, e.g., 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In some embodiments, at least one strand of the RNAi agent comprises a 5′ overhang of at least 1 nucleotide. In certain embodiments, at least one strand comprises a 5′ overhang of at least 2 nucleotides, e.g., 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In some embodiments, both the 3′ and the 5′ end of one strand of the RNAi agent comprise an overhang of at least 1 nucleotide. In some embodiments, at least one strand comprises a 3′ overhang of at least 2 nucleotides.

In certain embodiments, the double stranded RNAi agent further comprises a ligand. In certain embodiments, the ligand is conjugated to the 3′ end of the sense strand of the double stranded RNAi agent. In certain embodiments, the ligand is an N-acetylgalactosamine (GalNAc) derivative.

In certain embodiments, the ligand is

In certain embodiments, the wherein the double stranded RNAi agent is conjugated to the ligand as shown in the following schematic

and, wherein X is O or S.

In certain embodiments, the ligand is a cholesterol.

In certain embodiments, the ligand is one or more GalNAc derivatives attached through a monovalent, a bivalent, or a trivalent branched linker. The ligand may be conjugated to the 3′ end of the sense strand of the polynucleotide agent, the 5′ end of the sense strand of the polynucleotide agent, the 3′ end of the antisense strand of the polynucleotide agent, the 5′ end of the antisense strand of the polynucleotide agent.

In some embodiments, the polynucleotide agents of the invention comprise a plurality, e.g., 2, 3, 4, 5, or 6, of GalNAc, each independently attached to a plurality of nucleotides of the polynucleotide agent through a plurality of monovalent linkers.

In one embodiment, the agents inhibit the expression of GCK and the sense strand and the antisense strand comprise nucleotide sequences selected from the group consisting of the nucleotide sequences of any one of the agents listed in any one of Tables 2, 3, 6, and 7.

The invention provides cells containing the double stranded RNAi agents provided herein.

In another aspect, the invention provides pharmaceutical composition for inhibiting expression of a glucokinase (GCK) gene comprising the double stranded RNAi agent provided herein. In certain embodiments, the pharmaceutical compositions further comprise a lipid formulation.

The invention also provides methods of inhibiting expression of a glucokinase (GCK) gene in a cell. The methods include contacting the cell with a double stranded RNAi agent described herein; and maintaining the cell produced for a time sufficient to obtain degradation of the mRNA transcript of a GCK gene, thereby inhibiting expression of the GCK gene in the cell.

In certain embodiments, the cell is within a subject. In certain embodiments, the subject is a human. In certain embodiments, the human subject suffers from a disease or disorder that would benefit from reduction in GCK expression, such as a glycogen storage disease (GSD), e.g., type Ia GSD. In certain embodiments, the GCK expression is inhibited by at least about 5%, 10%, 15%, 20%, 25%, 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 98% or about 100%.

The invention further provides methods of treating a subject having a disorder that would benefit from reduction in expression of a glucokinase (GCK) gene. The methods include administering to the subject a therapeutically effective amount of any of the double stranded RNAi agents provided herein, thereby treating the subject, such as a disease or disorder associated with a glycogen storage disease (GSD), e.g., type Ia GSD.

In one aspect, the present invention provides methods of preventing at least one symptom in a subject having a disease or disorder that would benefit from reduction in expression of a glucokinase (GCK) gene. The methods include administering to the subject a prophylactically effective amount of a double stranded RNAi agent described herein, thereby preventing at least one symptom in the subject having a disorder that would benefit from reduction in GCK expression.

In certain embodiments, administration of the double stranded RNAi agent to the subject causes an increase in one or more blood glucose and/or a decrease in GCK protein accumulation. In certain embodiments, administration of the double stranded RNAi agent to the subject, e.g., a subject having a glycogen storage disease (GSD), e.g., type Ia GSD, causes a decrease in one or more signs of a glycogen storage disease (GSD), e.g., type Ia GSD, e.g., hypoglycemia, lactic acidosis, hyperuricemia, hyperlipidemia, hepatomegaly, kidney disease as a result of glycogen accumulation, hunger, jitteriness, lethargy, apnea, seizures, diaphoresis, confusion, headaches, dizziness, unusual mood or behavior changes, loss of consciousness, coma, muscle cramps, bleeding diathesis, short stauture, osteoporosis, delayed puberty, gout, renal disease, systemic hypertension, pulmonary hypertension, hepatic adenomas, pancreatitis, anemia, vitamin D deficiency, polycystic ovaries, irregular menstrual cycles, menorrhagia, and/or eruptive xanthomata.

The invention also provides methods of inhibiting the expression of a glucokinase (GCK) gene in a subject. The methods include method administering to the subject a therapeutically effective amount of any of the double stranded RNAi agents provided herein or a pharmaceutical composition comprising any of the double stranded RNAi agents provided herein, thereby inhibiting the expression of GCK in the subject.

In one aspect, the present invention provides methods of increasing the blood glucose levels, e.g., fasting blood glucose levels, in a subject having a disease or disorder that would benefit from reduction in GCK expression. The methods include administering to the subject a therapeutically effective amount of any of the double stranded RNAi agents provided herein or a pharmaceutical composition comprising any of the double stranded RNAi agents provided herein, thereby increasing the blood glucose levels in the subject.

In another aspect, the present invention provides methods of decreasing plasma lactate levels in a subject having a disease or disorder that would benefit from reduction in GCK expression. The methods include administering to the subject a therapeutically effective amount of any of the double stranded RNAi agents provided herein or a pharmaceutical composition comprising any of the double stranded RNAi agents provided herein, thereby decreasing the blood lactate levels in the subject.

In yet another aspect, the present invention provides methods of decreasing plasma uric acid levels in a subject having a disease or disorder that would benefit from reduction in GCK expression. The methods include administering to the subject a therapeutically effective amount of any of the double stranded RNAi agents provided herein or a pharmaceutical composition comprising any of the double stranded RNAi agents provided herein, thereby decreasing the plasma uric acidlevels in the subject.

In one aspect, the present invention provides methods of decreasing plasma triglyceride levels in a subject having a disease or disorder that would benefit from reduction in GCK expression. The methods include administering to the subject a therapeutically effective amount of any of the double stranded RNAi agents provided herein or a pharmaceutical composition comprising any of the double stranded RNAi agents provided herein, thereby decreasing plasma triglyceride levels in the subject.

In another aspect, the present invention provides methods of decreasing total plasma cholesterol levels in a subject having a disease or disorder that would benefit from reduction in GCK expression. The methods include administering to the subject a therapeutically effective amount of any of the double stranded RNAi agents provided herein or a pharmaceutical composition comprising any of the double stranded RNAi agents provided herein, thereby decreasing the total plasma cholesterol levels in said subject.

In one aspect, the present invention provides methods of decreasing hepatomegaly in a subject having a disease or disorder that would benefit from reduction in GCK expression. The methods include administering to the subject a therapeutically effective amount of any of the double stranded RNAi agents provided herein or a pharmaceutical composition comprising any of the double stranded RNAi agents provided herein, thereby decreasing hepatomegaly in the subject.

In certain embodiments, the dsRNA agent is administered to the subject at a dose of about 0.01 mg/kg to about 10 mg/kg or about 0.5 mg/kg to about 50 mg/kg. In certain embodiments, the dsRNA agent is administered about once per month, once every other two months, or once a quarter (i.e., once every three months), for example, at a dose of about 0.1 mg/kg to about 5.0 mg/kg.

In certain embodiments, the double stranded RNA agent is administered to the subject once a week. In certain embodiments, the dsRNA agent is administered to the subject once a month. In certain embodiments, the dsRNA agent is administered once per quarter (i.e., every three months).

In some embodiments, the methods of the invention further include administering an additional therapeutic to the subject. In one embodiment, the additional therepaeutic is a sodium-glucose co-transporter 2 (SGLT2) inhibitor, e.g., Dapagliflozin, Canagliflozin, Ipragliflozin (ASP-1941), Tofogliflozin, Empagliflozin, Sergliflozin etabonate, Remogliflozin etabonate (BHV091009), and Ertugliflozin (PF-04971729/MK-8835.

In yet another aspect, the invention provides kits for performing the methods of the invention. In one aspect, the invention provides a kit for performing a method for inhibiting expression of a glucokinase (GCK) gene in a cell by contacting a cell with a double stranded RNAi agent in an amount effective to inhibit expression of the GCK in the cell. The kit comprises an RNAi agent and instructions for use and, optionally, means for administering the RNAi agent to a subject.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides iRNA compositions, which affect the RNA-induced silencing complex (RISC)-mediated cleavage of RNA transcripts of a glucokinase (GCK) gene. The GCK gene may be within a cell, e.g., a cell within a subject, such as a human. The present invention also provides methods of using the iRNA compositions of the invention for inhibiting the expression of a glucokinase (GCK) gene and/or for treating a subject having a disorder that would benefit from inhibiting or reducing the expression of a GCK gene, such as a glycogen storage disease (GSD), e.g., type Ia GSD, and one or more of the signs or symptoms associated therewith.

The iRNAs of the invention include an RNA strand (the antisense strand) having a region which is about 30 nucleotides or less in length, e.g., 15-30, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides in length, which region is substantially complementary to at least part of an mRNA transcript of a GCK gene.

In certain embodiments, the iRNAs of the invention include an RNA strand (the antisense strand) which can include longer lengths, for example up to 66 nucleotides, e.g., 36-66, 26-36, 25-36, 31-60, 22-43, 27-53 nucleotides in length with a region of at least 19 contiguous nucleotides that is substantially complementary to at least a part of an mRNA transcript of a GCK gene. These iRNAs with the longer length antisense strands include a second RNA strand (the sense strand) of 20-60 nucleotides in length wherein the sense and antisense strands form a duplex of 18-30 contiguous nucleotides.

The use of these iRNAs enables the targeted degradation of mRNAs of a GCK gene in mammals. Very low dosages of GCK iRNAs, in particular, can specifically and efficiently mediate RNA interference (RNAi), resulting in significant inhibition of expression of a GCK gene. Using cell-based assays, the present inventors have demonstrated that iRNAs targeting GCK can mediate RNAi, resulting in significant inhibition of expression of a GCK gene. Thus, methods and compositions including these iRNAs are useful for treating a subject who would benefit by a reduction in the levels and/or activity of a GCK protein, such as a subject having a glycogen storage disease (GSD), e.g., type Ia GSD.

The following detailed description discloses how to make and use compositions containing iRNAs to inhibit the expression of a GCK gene, as well as compositions and methods for treating subjects having diseases and disorders that would benefit from inhibition and/or reduction of the expression of this gene.

I. Definitions

In order that the present invention may be more readily understood, certain terms are first defined. In addition, it should be noted that whenever a value or range of values of a parameter are recited, it is intended that values and ranges intermediate to the recited values are also intended to be part of this invention.

The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element, e.g., a plurality of elements.

The term “including” is used herein to mean, and is used interchangeably with, the phrase “including but not limited to”. The term “or” is used herein to mean, and is used interchangeably with, the term “and/or,” unless context clearly indicates otherwise.

The term “about” is used herein to mean within the typical ranges of tolerances in the art. For example, “about” can be understood as within about 2 standard deviations from the mean. In certain embodiments, about means +10%. In certain embodiments, about means +5%. When about is present before a series of numbers or a range, it is understood that “about” can modify each of the numbers in the series or range.

The terms “GCK,” “glucokinase,” “hexokinase D,” and “hexokinase 4” refer to an enzyme that facilitates phosphorylation of glucose to glucose-6-phosphate having an amino acid sequence from any vertebrate or mammalian source, including, but not limited to, human, bovine, chicken, rodent, mouse, rat, porcine, ovine, primate, monkey, and guinea pig, unless specified otherwise. The term also refers to fragments and variants of native GCK that maintain at least one in vivo or in vitro activity of a native GCK. The term encompasses full-length unprocessed precursor forms of GCK as well as mature forms resulting from, e.g., post-translational processing.

The sequence of a human GCK mRNA transcript (transcript variant 2) can be found at, for example, GenBank Accession No. GI: 15967158 (NM_033507; NCBI GeneID: 2645; SEQ ID NO:1). The sequence of another human GCK mRNA transcript (transcript variant 1) can be found at, for example, GenBank Accession No. GI: 167621407 (NM_000162); SEQ ID NO:2). The sequence of yet another human GCK mRNA transcript (transcript variant 3) can be found at, for example, GenBank Accession No. GI: 15967160 (NM_033508); SEQ ID NO:3). The predicted sequence of a rhesus GCK mRNA transcript (transcript variant X1) can be found at, for example, GenBank Accession No. GI: 544420246 (XM_005549685; SEQ ID NO:4). The predicted sequence of another rhesus GCK mRNA transcript (transcript variant X2) can be found at, for example, GenBank Accession No. GI: 544420248 (XM_005549686; SEQ ID NO:5). The predicted sequence of yet another rhesus GCK mRNA transcript (transcript variant X3) can be found at, for example, GenBank Accession No. GI: 544420250 (XM_005549687; SEQ ID NO:6). The predicted sequence of another rhesus GCK mRNA transcript (transcript variant X4) can be found at, for example, GenBank Accession No. GI: 544420252 (XM_005549688; SEQ ID NO:7). The sequence of a mouse GCK mRNA transcript (transcript variant 1) can be found at, for example, GenBank Accession No. GI: 565671706 (NM_010292; SEQ ID NO:8). The sequence of another mouse GCK mRNA transcript (transcript variant 2) can be found at, for example, GenBank Accession No. GI: 565671714 (NM_001287386; SEQ ID NO:9). The sequence of a rat GCK mRNA transcript (transcript variant 2) can be found at, for example, GenBank Accession No. GI: 399220372 (NM_012565; SEQ ID NO:10). The sequence of another rat GCK mRNA transcript (transcript variant 1) can be found at, for example, GenBank Accession No. GI: 399220370 (NM_001270849; SEQ ID NO:11). The sequence of yet another rat GCK mRNA transcript (transcript variant 3) can be found at, for example, GenBank Accession No. GI: 399220373 (NM_001270850; SEQ ID NO:12). Additional examples of GCK mRNA sequences are readily available using publicly available databases, e.g., GenBank, UniProt, and OMIM.

As used herein, “target sequence” refers to a contiguous portion of the nucleotide sequence of an mRNA molecule formed during the transcription of a GCK gene, including mRNA that is a product of RNA processing of a primary transcription product. In one embodiment, the target portion of the sequence will be at least long enough to serve as a substrate for iRNA-directed cleavage at or near that portion of the nucleotide sequence of an mRNA molecule formed during the transcription of a GCK gene.

The target sequence may be from about 9-36 nucleotides in length, e.g., about 15-30 nucleotides in length. For example, the target sequence can be from about 15-30 nucleotides, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides in length. Ranges and lengths intermediate to the above recited ranges and lengths are also contemplated to be part of the invention.

As used herein, the term “strand comprising a sequence” refers to an oligonucleotide comprising a chain of nucleotides that is described by the sequence referred to using the standard nucleotide nomenclature.

“G,” “C,” “A,” “T,” and “U” each generally stand for a nucleotide that contains guanine, cytosine, adenine, thymidine, and uracil as a base, respectively. However, it will be understood that the term “ribonucleotide” or “nucleotide” can also refer to a modified nucleotide, as further detailed below, or a surrogate replacement moiety. The skilled person is well aware that guanine, cytosine, adenine, and uracil can be replaced by other moieties without substantially altering the base pairing properties of an oligonucleotide comprising a nucleotide bearing such replacement moiety. For example, without limitation, a nucleotide comprising inosine as its base can base pair with nucleotides containing adenine, cytosine, or uracil. Hence, nucleotides containing uracil, guanine, or adenine can be replaced in the nucleotide sequences of dsRNA featured in the invention by a nucleotide containing, for example, inosine. In another example, adenine and cytosine anywhere in the oligonucleotide can be replaced with guanine and uracil, respectively to form G-U Wobble base pairing with the target mRNA. Sequences containing such replacement moieties are suitable for the compositions and methods featured in the invention.

The terms “iRNA,” “RNAi agent,” “iRNA agent,” and “RNA interference agent” as used interchangeably herein, refer to an agent that contains RNA as that term is defined herein, and which mediates the targeted cleavage of an RNA transcript via an RNA-induced silencing complex (RISC) pathway. iRNA directs the sequence-specific degradation of mRNA through a process known as RNA interference (RNAi). The iRNA modulates, e.g., inhibits, the expression of GCK in a cell, e.g., a cell within a subject, such as a mammalian subject.

In one embodiment, an RNAi agent of the invention includes a single stranded RNAi that interacts with a target RNA sequence, e.g., a GCK target mRNA sequence, to direct the cleavage of the target RNA. Without wishing to be bound by theory it is believed that long double stranded RNA introduced into cells is broken down into double stranded short interfering RNAs (siRNAs) comprising a sense strand and an antisense strand by a Type III endonuclease known as Dicer (Sharp et al. (2001) Genes Dev. 15:485). Dicer, a ribonuclease-III-like enzyme, processes these dsRNA into 19-23 base pair short interfering RNAs with characteristic two base 3′ overhangs (Bernstein, et al., (2001) Nature 409:363). These siRNAs are then incorporated into an RNA-induced silencing complex (RISC) where one or more helicases unwind the siRNA duplex, enabling the complementary antisense strand to guide target recognition (Nykanen, et al., (2001) Cell 107:309). Upon binding to the appropriate target mRNA, one or more endonucleases within the RISC cleave the target to induce silencing (Elbashir, et al., (2001) Genes Dev. 15:188). Thus, in one aspect the invention relates to a single stranded RNA (ssRNA) (the antisense strand of an siRNA duplex) generated within a cell and which promotes the formation of a RISC complex to effect silencing of the target gene, i.e., a GCK gene. Accordingly, the term “siRNA” is also used herein to refer to an RNAi as described above.

In another embodiment, the RNAi agent may be a single-stranded RNA that is introduced into a cell or organism to inhibit a target mRNA. Single-stranded RNAi agents bind to the RISC endonuclease, Argonaute 2, which then cleaves the target mRNA. The single-stranded siRNAs are generally 15-30 nucleotides and are chemically modified. The design and testing of single-stranded RNAs are described in U.S. Pat. No. 8,101,348 and in Lima et al., (2012) Cell 150:883-894, the entire contents of each of which are hereby incorporated herein by reference. Any of the antisense nucleotide sequences described herein may be used as a single-stranded siRNA as described herein or as chemically modified by the methods described in Lima et al., (2012) Cell 150:883-894.

In another embodiment, an “iRNA” for use in the compositions and methods of the invention is a double stranded RNA and is referred to herein as a “double stranded RNAi agent,” “double stranded RNA (dsRNA) molecule,” “dsRNA agent,” or “dsRNA”. The term “dsRNA”, refers to a complex of ribonucleic acid molecules, having a duplex structure comprising two anti-parallel and substantially complementary nucleic acid strands, referred to as having “sense” and “antisense” orientations with respect to a target RNA, i.e., a GCK gene. In some embodiments of the invention, a double stranded RNA (dsRNA) triggers the degradation of a target RNA, e.g., an mRNA, through a post-transcriptional gene-silencing mechanism referred to herein as RNA interference or RNAi.

The majority of nucleotides of each strand of a dsRNA molecule may be ribonucleotides, but as described in detail herein, each or both strands can also include one or more non-ribonucleotides, e.g., a deoxyribonucleotide and/or a modified nucleotide. In addition, as used in this specification, an “RNAi agent” may include ribonucleotides with chemical modifications; an RNAi agent may include substantial modifications at multiple nucleotides. As used herein, the term “modified nucleotide” refers to a nucleotide having, independently, a modified sugar moiety, a modified internucleotide linkage, and/or modified nucleobase. Thus, the term modified nucleotide encompasses substitutions, additions or removal of, e.g., a functional group or atom, to internucleoside linkages, sugar moieties, or nucleobases. The modifications suitable for use in the agents of the invention include all types of modifications disclosed herein or known in the art. Any such modifications, as used in a siRNA type molecule, are encompassed by “RNAi agent” for the purposes of this specification and claims.

The duplex region may be of any length that permits specific degradation of a desired target RNA through a RISC pathway, and may range from about 9 to 36 base pairs in length, e.g., about 15-30 base pairs in length, for example, about 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 base pairs in length, such as about 15-30, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs in length. Ranges and lengths intermediate to the above recited ranges and lengths are also contemplated to be part of the invention.

The two strands forming the duplex structure may be different portions of one larger RNA molecule, or they may be separate RNA molecules. Where the two strands are part of one larger molecule, and therefore are connected by an uninterrupted chain of nucleotides between the 3′-end of one strand and the 5′-end of the respective other strand forming the duplex structure, the connecting RNA chain is referred to as a “hairpin loop.” A hairpin loop can comprise at least one unpaired nucleotide. In some embodiments, the hairpin loop can comprise at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 23, or more unpaired nucleotides. In some embodiments, the hairpin loop can be 10 or fewer nucleotides. In some embodiments, the hairpin loop can be 8 or fewer unpaired nucleotides. In some embodiments, the hairpin loop can be 4-10 unpaired nucleotides. In some embodiments, the hairpin loop can be 4-8 nucleotides.

Where the two substantially complementary strands of a dsRNA are comprised by separate RNA molecules, those molecules need not, but can be covalently connected. Where the two strands are connected covalently by means other than an uninterrupted chain of nucleotides between the 3′-end of one strand and the 5′-end of the respective other strand forming the duplex structure, the connecting structure is referred to as a “linker.” The RNA strands may have the same or a different number of nucleotides. The maximum number of base pairs is the number of nucleotides in the shortest strand of the dsRNA minus any overhangs that are present in the duplex. In addition to the duplex structure, an RNAi may comprise one or more nucleotide overhangs.

In one embodiment, an RNAi agent of the invention is a dsRNA, each strand of which comprises 19-23 nucleotides, that interacts with a target RNA sequence, e.g., a GCK gene, without wishing to be bound by theory, long double stranded RNA introduced into cells is broken down into siRNA by a Type III endonuclease known as Dicer (Sharp et al. (2001) Genes Dev. 15:485). Dicer, a ribonuclease-III-like enzyme, processes the dsRNA into 19-23 base pair short interfering RNAs with characteristic two base 3′ overhangs (Bernstein, et al., (2001) Nature 409:363). The siRNAs are then incorporated into an RNA-induced silencing complex (RISC) where one or more helicases unwind the siRNA duplex, enabling the complementary antisense strand to guide target recognition (Nykanen, et al., (2001) Cell 107:309). Upon binding to the appropriate target mRNA, one or more endonucleases within the RISC cleave the target to induce silencing (Elbashir, et al., (2001) Genes Dev. 15:188). In one embodiment, an RNAi agent of the invention is a dsRNA of 24-30 nucleotides that interacts with a target RNA sequence, e.g., a prolyl hydroxylase domain-containing gene, i.e., a PHD1 target mRNA sequence, a PHD2 target mRNA sequence, or a PHD3 target mRNA sequence, to direct the cleavage of the target RNA.

As used herein, the term “nucleotide overhang” refers to at least one unpaired nucleotide that protrudes from the duplex structure of an iRNA, e.g., a dsRNA. For example, when a 3′-end of one strand of a dsRNA extends beyond the 5′-end of the other strand, or vice versa, there is a nucleotide overhang. A dsRNA can comprise an overhang of at least one nucleotide; alternatively the overhang can comprise at least two nucleotides, at least three nucleotides, at least four nucleotides, at least five nucleotides or more. A nucleotide overhang can comprise or consist of a nucleotide/nucleoside analog, including a deoxynucleotide/nucleoside. The overhang(s) can be on the sense strand, the antisense strand or any combination thereof. Furthermore, the nucleotide(s) of an overhang can be present on the 5′-end, 3′-end or both ends of either an antisense or sense strand of a dsRNA.

In one embodiment of the dsRNA, at least one strand comprises a 3′ overhang of at least 1 nucleotide. In another embodiment, at least one strand comprises a 3′ overhang of at least 2 nucleotides, e.g., 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In other embodiments, at least one strand of the RNAi agent comprises a 5′ overhang of at least 1 nucleotide. In certain embodiments, at least one strand comprises a 5′ overhang of at least 2 nucleotides, e.g., 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In still other embodiments, both the 3′ and the 5′ end of one strand of the RNAi agent comprise an overhang of at least 1 nucleotide.

In one embodiment, the antisense strand of a dsRNA has a 1-10 nucleotide, e.g., a 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide, overhang at the 3′-end and/or the 5′-end. In one embodiment, the sense strand of a dsRNA has a 1-10 nucleotide, e.g., a 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide, overhang at the 3′-end and/or the 5′-end. In certain embodiments, the overhang on the sense strand or the antisense strand, or both, can include extended lengths longer than 10 nucleotides, e.g., 1-30 nucleotides, 2-30 nucleotides, 10-30 nucleotides, or 10-15 nucleotides in length. In certain embodiments, an extended overhang is on the sense strand of the duplex. In certain embodiments, an extended overhang is present on the 3′end of the sense strand of the duplex. In certain embodiments, an extended overhang is present on the 5′end of the sense strand of the duplex. In certain embodiments, an extended overhang is on the antisense strand of the duplex. In certain embodiments, an extended overhang is present on the 3′end of the antisense strand of the duplex. In certain embodiments, an extended overhang is present on the 5′end of the antisense strand of the duplex. In another embodiment, one or more of the nucleotides in the overhang is replaced with a nucleoside thiophosphate.

The terms “blunt” or “blunt ended” as used herein in reference to a dsRNA mean that there are no unpaired nucleotides or nucleotide analogs at a given terminal end of a dsRNA, i.e., no nucleotide overhang. One or both ends of a dsRNA can be blunt. Where both ends of a dsRNA are blunt, the dsRNA is said to be blunt ended. To be clear, a “blunt ended” dsRNA is a dsRNA that is blunt at both ends, i.e., no nucleotide overhang at either end of the molecule. Most often such a molecule will be double stranded over its entire length.

The term “antisense strand” or “guide strand” refers to the strand of an iRNA, e.g., a dsRNA, which includes a region that is substantially complementary to a target sequence, e.g., a GCK mRNA. As used herein, the term “region of complementarity” refers to the region on the antisense strand that is substantially complementary to a sequence, for example a target sequence, e.g., a GCK nucleotide sequence, as defined herein. Where the region of complementarity is not fully complementary to the target sequence, the mismatches can be in the internal or terminal regions of the molecule. Generally, the most tolerated mismatches are in the terminal regions, e.g., within 5, 4, 3, or 2 nucleotides of the 5′- and/or 3′-terminus of the iRNA.

The term “sense strand” or “passenger strand” as used herein, refers to the strand of an iRNA that includes a region that is substantially complementary to a region of the antisense strand as that term is defined herein.

As used herein, the term “cleavage region” refers to a region that is located immediately adjacent to the cleavage site. The cleavage site is the site on the target at which cleavage occurs. In some embodiments, the cleavage region comprises three bases on either end of, and immediately adjacent to, the cleavage site. In some embodiments, the cleavage region comprises two bases on either end of, and immediately adjacent to, the cleavage site. In some embodiments, the cleavage site specifically occurs at the site bound by nucleotides 10 and 11 of the antisense strand, and the cleavage region comprises nucleotides 11, 12 and 13.

As used herein, and unless otherwise indicated, the term “complementary,” when used to describe a first nucleotide sequence in relation to a second nucleotide sequence, refers to the ability of an oligonucleotide or polynucleotide comprising the first nucleotide sequence to hybridize and form a duplex structure under certain conditions with an oligonucleotide or polynucleotide comprising the second nucleotide sequence, as will be understood by the skilled person. Such conditions can, for example, be stringent conditions, where stringent conditions can include: 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50° C. or 70° C. for 12-16 hours followed by washing (see, e.g., “Molecular Cloning: A Laboratory Manual, Sambrook, et al. (1989) Cold Spring Harbor Laboratory Press). Other conditions, such as physiologically relevant conditions as can be encountered inside an organism, can apply. The skilled person will be able to determine the set of conditions most appropriate for a test of complementarity of two sequences in accordance with the ultimate application of the hybridized nucleotides.

Complementary sequences within an iRNA, e.g., within a dsRNA as described herein, include base-pairing of the oligonucleotide or polynucleotide comprising a first nucleotide sequence to an oligonucleotide or polynucleotide comprising a second nucleotide sequence over the entire length of one or both nucleotide sequences. Such sequences can be referred to as “fully complementary” with respect to each other herein. However, where a first sequence is referred to as “substantially complementary” with respect to a second sequence herein, the two sequences can be fully complementary, or they can form one or more, but generally not more than 5, 4, 3, or 2 mismatched base pairs upon hybridization for a duplex up to 30 base pairs, while retaining the ability to hybridize under the conditions most relevant to their ultimate application, e.g., inhibition of gene expression via a RISC pathway. However, where two oligonucleotides are designed to form, upon hybridization, one or more single stranded overhangs, such overhangs shall not be regarded as mismatches with regard to the determination of complementarity. For example, a dsRNA comprising one oligonucleotide 21 nucleotides in length and another oligonucleotide 23 nucleotides in length, wherein the longer oligonucleotide comprises a sequence of 21 nucleotides that is fully complementary to the shorter oligonucleotide, can yet be referred to as “fully complementary” for the purposes described herein.

“Complementary” sequences, as used herein, can also include, or be formed entirely from, non-Watson-Crick base pairs and/or base pairs formed from non-natural and modified nucleotides, in so far as the above requirements with respect to their ability to hybridize are fulfilled. Such non-Watson-Crick base pairs include, but are not limited to, G:U Wobble or Hoogstein base pairing.

The terms “complementary,” “fully complementary” and “substantially complementary” herein can be used with respect to the base matching between the sense strand and the antisense strand of a dsRNA, or between the antisense strand of an iRNA agent and a target sequence, as will be understood from the context of their use.

As used herein, a polynucleotide that is “substantially complementary to at least part of” a messenger RNA (mRNA) refers to a polynucleotide that is substantially complementary to a contiguous portion of the mRNA of interest (e.g., an mRNA encoding GCK). For example, a polynucleotide is complementary to at least a part of a GCK mRNA if the sequence is substantially complementary to a non-interrupted portion of an mRNA encoding GCK.

Accordingly, in some embodiments, the antisense strand polynucleotides disclosed herein are fully complementary to the target GCK sequence. In other embodiments, the antisense strand polynucleotides disclosed herein are substantially complementary to the target GCK sequence and comprise a contiguous nucleotide sequence which is at least about 80% complementary over its entire length to the equivalent region of the nucleotide sequence of SEQ ID NO:1, or a fragment of SEQ ID NO:1, such as about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about % 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% complementary.

In other embodiments, the antisense polynucleotides disclosed herein are substantially complementary to the target GCK sequence and comprise a contiguous nucleotide sequence which is at least about 80% complementary over its entire length to any one of the sense strand nucleotide sequences in any one of Tables 2, 3, 6, and 7, or a fragment of any one of the sense strand nucleotide sequences in any one of Tables 2, 3, 6, and 7, such as at least 85%, 90%, 95% complementary, or 100% complementary.

In one embodiment, an RNAi agent of the invention includes a sense strand that is substantially complementary to an antisense polynucleotide which, in turn, is complementary to a target GCK sequence, and wherein the sense strand polynucleotide comprises a contiguous nucleotide sequence which is at least about 80% complementary over its entire length to the equivalent region of the nucleotide sequence of SEQ ID NO:5, or a fragment of any one of SEQ ID NO:5, such as about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about % 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% complementary.

The term “inhibiting,” as used herein, is used interchangeably with “reducing,” “silencing,” “downregulating,” “suppressing” and other similar terms, and includes any level of inhibition.

The phrase “inhibiting expression of a GCK,” as used herein, includes inhibition of expression of any GCK gene (such as, e.g., a mouse GCK gene, a rat GCK gene, a monkey GCK gene, or a human GCK gene) as well as variants or mutants of a GCK gene that encode a GCK protein.

“Inhibiting expression of a GCK gene” includes any level of inhibition of a GCK gene, e.g., at least partial suppression of the expression of a GCK gene, such as an inhibition by at least about 20%. In certain embodiments, inhibition is by at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%.

The expression of a GCK gene may be assessed based on the level of any variable associated with GCK gene expression, e.g., GCK mRNA level or GCK protein level. The expression of a GCK may also be assessed indirectly based on, e.g., blood glucose levels, serum ketone body levels, and/or serum fatty acid levels. Inhibition may be assessed by a change in an absolute or relative level of one or more of these variables compared with a control level. The control level may be any type of control level that is utilized in the art, e.g., a pre-dose baseline level, or a level determined from a similar subject, cell, or sample that is untreated or treated with a control (such as, e.g., buffer only control or inactive agent control).

In one embodiment, at least partial suppression of the expression of a GCK gene, is assessed by a reduction of the amount of GCK mRNA which can be isolated from or detected in a first cell or group of cells in which a GCK gene is transcribed and which has or have been treated such that the expression of a GCK gene is inhibited, as compared to a second cell or group of cells substantially identical to the first cell or group of cells but which has or have not been so treated (control cells). The degree of inhibition may be expressed in terms of:

${\frac{\left( {{mRNA}\mspace{14mu}{in}\mspace{14mu}{control}\mspace{14mu}{cells}} \right) - \left( {{mRNA}\mspace{14mu}{in}\mspace{14mu}{treated}\mspace{11mu}{cells}} \right)}{\left( {{mRNA}\mspace{14mu}{in}\mspace{14mu}{control}\mspace{14mu}{cells}} \right)} \cdot 100}\%$

The phrase “contacting a cell with an RNAi agent,” such as a dsRNA, as used herein, includes contacting a cell by any possible means. Contacting a cell with an RNAi agent includes contacting a cell in vitro with the iRNA or contacting a cell in vivo with the iRNA. The contacting may be done directly or indirectly. Thus, for example, the RNAi agent may be put into physical contact with the cell by the individual performing the method, or alternatively, the RNAi agent may be put into a situation that will permit or cause it to subsequently come into contact with the cell.

Contacting a cell in vitro may be done, for example, by incubating the cell with the RNAi agent. Contacting a cell in vivo may be done, for example, by injecting the RNAi agent into or near the tissue where the cell is located, or by injecting the RNAi agent into another area, e.g., the bloodstream or the subcutaneous space, such that the agent will subsequently reach the tissue where the cell to be contacted is located. For example, the RNAi agent may contain and/or be coupled to a ligand, e.g., GalNAc3, that directs the RNAi agent to a site of interest, e.g., the liver. Combinations of in vitro and in vivo methods of contacting are also possible. For example, a cell may also be contacted in vitro with an RNAi agent and subsequently transplanted into a subject.

In one embodiment, contacting a cell with an iRNA includes “introducing” or “delivering the iRNA into the cell” by facilitating or effecting uptake or absorption into the cell. Absorption or uptake of an iRNA can occur through unaided diffusive or active cellular processes, or by auxiliary agents or devices. Introducing an iRNA into a cell may be in vitro and/or in vivo. For example, for in vivo introduction, iRNA can be injected into a tissue site or administered systemically. In vivo delivery can also be done by a beta-glucan delivery system, such as those described in U.S. Pat. Nos. 5,032,401 and 5,607,677, and U.S. Publication No. 2005/0281781, the entire contents of which are hereby incorporated herein by reference. In vitro introduction into a cell includes methods known in the art such as electroporation and lipofection. Further approaches are described herein below and/or are known in the art.

The term “lipid nanoparticle” or “LNP” is a vesicle comprising a lipid layer encapsulating a pharmaceutically active molecule, such as a nucleic acid molecule, e.g., an iRNA or a plasmid from which an iRNA is transcribed. LNPs are described in, for example, U.S. Pat. Nos. 6,858,225, 6,815,432, 8,158,601, and 8,058,069, the entire contents of which are hereby incorporated herein by reference.

As used herein, a “subject” is an animal, such as a mammal, including a primate (such as a human, a non-human primate, e.g., a monkey, and a chimpanzee), a non-primate (such as a cow, a pig, a camel, a llama, a horse, a goat, a rabbit, a sheep, a hamster, a guinea pig, a cat, a dog, a rat, a mouse, a horse, and a whale), or a bird (e.g., a duck or a goose). It is understood that the sequence of the GCK gene must be sufficiently complementary to the antisense strand of the iRNA agent for the agent to be used in the indicated species.

In an embodiment, the subject is a human, such as a human being treated or assessed for a disease, disorder or condition that would benefit from reduction in GCK expression; a human at risk for a disease, disorder or condition that would benefit from reduction in GCK expression; a human having a disease, disorder or condition that would benefit from reduction in GCK expression; and/or human being treated for a disease, disorder or condition that would benefit from reduction in GCK expression as described herein.

As used herein, the terms “treating” or “treatment” refer to a beneficial or desired result including, such as increasing blood glucose levels in a subject. The terms “treating” or “treatment” also include, but are not limited to, alleviation or amelioration of one or more symptoms of a glycogen storage disease (GSD), e.g., type Ia GSD, or at least one sign or symptom associated with GSD, such as hypoglycemia, lactic acidosis, hyperuricemia, hyperlipidemia, hepatomegaly, kidney disease as a result of glycogen accumulation, hunger, jitteriness, lethargy, apnea, seizures, diaphoresis, confusion, headaches, dizziness, unusual mood or behavior changes, loss of consciousness, coma, muscle cramps, bleeding diathesis, short stauture, osteoporosis, delayed puberty, gout, renal disease, systemic hypertension, pulmonary hypertension, hepatic adenomas, pancreatitis, anemia, vitamin D deficiency, polycystic ovaries, irregular menstrual cycles, menorrhagia, and eruptive xanthomata.

“Treatment” can also mean prolonging survival as compared to expected survival in the absence of treatment.

By “lower” in the context of a disease marker or symptom is meant a statistically significant decrease in such level. The decrease can be, for example, at least 10%, at least 20%, at least 30%, at least 40%, or more, down to a level accepted as within the range of normal for an individual without such disorder, or to below the level of detection of the assay. In certain embodiments, the decrease is down to a level accepted as within the range of normal for an individual without such disorder which can also be referred to as a normalization of a level. For example, lowering cholesterol to 180 mg/dl or lower would be considered to be within the range of normal for a subject. A subject having a cholesterol level of 230 mg/dl with a cholesterol level decreased to 210 mg/dl would have a cholesterol level that was decreased by 40% towards normal (230−210/230−180=20/50=40% reduction). In certain embodiments, the reduction is the normalization of the level of a sign or symptom of a disease, a reduction in the difference between the subject level of a sign of the disease and the normal level of the sign for the disease (e.g., to the upper level of normal when the value for the subject must be decreased to reach a normal value, and to the lower level of normal when the value for the subject must be increased to reach a normal level). In certain embodiments, the methods include a clinically relevant inhibition of expression of a GCK gene, e.g. as demonstrated by a clinically relevant outcome after treatment of a subject with an agent to reduce the expression of a GCK gene.

As used herein, “prevention” or “preventing,” when used in reference to a disease, disorder or condition thereof, that would benefit from a reduction in expression of a GCK gene, refers to a reduction in the likelihood that a subject will develop a symptom associated with such disease, disorder, or condition, e.g., a glycogen storage disease (GSD), e.g., type Ia GSD. The likelihood of developing type Ia GSD is reduced, for example, when an individual having one or more risk factors for type Ia GSD, e.g., a genetic disorder, either fails to develop type Ia GSD, or signs or symptoms thereof, or develops type Ia GSD, or signs or symptoms thereof, with less severity relative to a population having the same risk factors and not receiving treatment as described herein.

The failure to develop a disease, disorder or condition, or the reduction in the development of a symptom associated with such a disease, disorder or condition (e.g., by at least about 10% on a clinically accepted scale for that disease or disorder), or the exhibition of delayed symptoms delayed (e.g., by days, weeks, months, or years) is considered effective prevention. Prevention can require administration of more than one dose of an agent described herein.

As used herein, the term “blood glucose level” refers to the level of glucose present in blood as determined by any routine method known in the art. It is understood that the glucose level in a subject sample is dependent on, for example, whether the subject has had a meal, whether the subject has fasted, the time of day and the level of activity of the subject. Therefore, the glucose level must be compared to an appropriate control to determine if the glucose level is, in fact, altered from a normal level or from a level obtained from the subject at an earlier time point, e.g., prior to treatment. In general, blood glucose levels, e.g., fasting blood glucose levels, in a subject that does not have a disease or disorder that would benefit from reduction in the levels of GCK as described herein is about 70 to about 120 mg/dL.

As used herein, the term “plasma lactate level” refers to the level of lactate present in blood as determined by any routine method known in the art. It is understood that the lactate level in a subject sample is dependent on, for example, whether the subject has fasted, the time of day and the level of activity of the subject. Therefore, the lactate level must be compared to an appropriate control to determine if the lactate level is, in fact, altered from a normal level or from a level obtained from the subject at an earlier time point, e.g., prior to treatment. In general, plasma lactate levels in a subject that does not have a disease or disorder that would benefit from reduction in the levels of GCK as described herein is about 0.5 to about 2.2 mmol/L.

As used herein, the term “plasma uric acid level” refers to the level of uric acid present in blood as determined by any routine method known in the art. It is understood that the lactate level in a subject sample is dependent on, for example, whether the subject has fasted, the time of day and the level of activity of the subject. Therefore, the uric acid level must be compared to an appropriate control to determine if the uric acid level is, in fact, altered from a normal level or from a level obtained from the subject at an earlier time point, e.g., prior to treatment. In general, plasma uric acid levels in a subject that does not have a disease or disorder that would benefit from reduction in the levels of GCK as described herein is about 2.0 to about 5.0 mg/dL.

As used herein, the term “plasma triglyceride level” refers to the level of triglycerides present in blood as determined by any routine method known in the art. It is understood that the triglyceride level in a subject sample is dependent on, for example, whether the subject has fasted, the time of day and the level of activity of the subject.

Therefore, the triglyceride level must be compared to an appropriate control to determine if the triglyceride level is, in fact, altered from a normal level or from a level obtained from the subject at an earlier time point, e.g., prior to treatment. In general, plasma triglyceride levels in a subject that does not have a disease or disorder that would benefit from reduction in the levels of GCK as described herein is about 150 to about 200 mg/dL.

As used herein, the term “total cholesterol level” refers to the level of high density lipoprotein (HDL) plus low density lipoprotein (LDL) plus 20% of the triglyceride level as determined by any routine method known in the art. It is understood that the total cholesterol level in a subject sample is dependent on, for example, whether the subject has fasted, the time of day and the level of activity of the subject. Therefore, the total cholesterol level must be compared to an appropriate control to determine if the total cholesterol level is, in fact, altered from a normal level or from a level obtained from the subject at an earlier time point, e.g., prior to treatment. In general, plasma cholesterol levels in a subject that does not have a disease or disorder that would benefit from reduction in the levels of GCK as described herein is about 100 to about 200 mg/dL.

As used herein, the term “hepatomegaly” refers to swelling of the liver beyond its normal size as determined by any routine method known in the art. It is understood that the size and weight of the liver in a subject that does not have a disease or disorder that would benefit from reduction in the levels of GCK as described herein increases with age and body weight. Sex and body shape also influence the size of the liver, e.g., by percussion, the mean liver size is about 7.5 centimeters in adult women and about 10.5 centimeters in adult men; it may be about 3 centimeters larger or smaller and still be normal.

As used herein, a “disease or disorder that would benefit from reduction in GCK expression” is a disease or disorder associated with or caused by a clinically relevant hypoglycemia. For example, this term includes any disorder, disease or condition resulting in one or more signs or symptoms of a glycogen storage disease (GSD), e.g., type Ia GSD, including, but not limited to, hypoglycemia, lactic acidosis, hyperuricemia, hyperlipidemia, hepatomegaly, kidney disease as a result of glycogen accumulation, hunger, jitteriness, lethargy, apnea, seizures, diaphoresis, confusion, headaches, dizziness, unusual mood or behavior changes, loss of consciousness, coma, muscle cramps, bleeding diathesis, short stauture, osteoporosis, delayed puberty, gout, renal disease, systemic hypertension, pulmonary hypertension, hepatic adenomas, pancreatitis, anemia, vitamin D deficiency, polycystic ovaries, irregular menstrual cycles, menorrhagia, and eruptive xanthomata. In certain embodiments, a “disease or disorder that would benefit from reduction in GCK expression” meets the diagnostic requirements of a type Ia GSD.

“Glycogen storage disease” or “GSD” is an inherited genetic disorder due to an absence or deficiency of one of the enzymes responsible for making or breaking down glycogen in the body. This enzyme deficiency causes either abnormal tissue concentrations of glycogen or incorrectly or abnormally formed glycogen. There are about 11 known types of GSD, which are classified based on the missing or defective enzymes. For example, Type Ia GSD, the most common form of GSD, is caused by a genetic defect in the enzyme glucose-6-phosphatase. Hepatomegaly due to inappropriate glycogen accumulation and various metabolic disarrangements from inappropriate glucose-6-phosphate metabolism are predominant features of many of the various glycogen storage diseases, such as Types Ia, Ib, III, IV, VI and IX. Symptoms of GSD vary based on the enzyme that is missing and usually result from the buildup of glycogen or from the inability to produce glucose when needed. Glycogen storage disease is usually diagnosed in infancy or childhood, e.g., at about age 3-4. Affected children typically present with hepatomegaly, lactic acidosis, hyperuricemia, hyperlipidemia, hypertriglyceridemia and/or hypoglycemic seizures. Further, affected children often have doll-like faces with fat cheeks, relatively thin extremities, short stature, and protuberant abdomen. Xanthoma and diarrhea may be present. Impaired platelet function can lead to a bleeding tendency with frequent epistaxis. The diagnostic criteria for GSD, e.g., type Ia GSD, include, for example, fasting blood glucose concentration lower than 60 mg/dL; plasma lactate higher than 2.5 mmol/L; plasma uric acid higher than 5.0 mg/dL; triglycerides higher than 250 mg/dL; total plasma cholesterol higher than 200 mg/dL; administration of glucagon or epinephrine (i.e., a glucagon or epinephrine challenge test) causes little or no increase in blood glucose concentration, but both increase serum lactate concentrations significantly; histopathologic liver findings which include distention of the liver cells by glycogen and fat; PAS positive and diastase sensitive glycogen that is uniformly distributed within the cytoplasm; normal or only modestly increased glycogen; and large and numerous lipid vacuoles; biallelic mutations in either G6PC (GSDIa) or SLC37A4 (GSDIb) (Veiga-da-Cunha et al (1998) Am J Hum Genet. 63:976-83; Chou et al (2002) Hum Mutat. 29:921-30; Matern et al (2002) Eur J Pediatr. 161 Suppl 1:S10-9; Rake et al (2002) Eur J Pediatr. 161 Suppl 1:S20-34; and Ekstein et al (2004) Am J Med Genet. 129A:162-4); deficient, e.g., (lower than 10% of normal) glucose-6-phosphatase (G6Pase) catalytic activity (the normal G6Pase enzyme activity level in liver is 3.50±0.8 μmol/min/g tissue, although in rare individuals with milder clinical manifestations, the G6Pase enzyme activity can be higher, e.g., >1.0 μmol/min/g tissue and <2.0 μmol/min/g tissue.

“Therapeutically effective amount,” as used herein, is intended to include the amount of an RNAi agent that, when administered to a subject having a glycogen storage disease (GSD), e.g., type Ia GSD, is sufficient to effect treatment of the disease (e.g., by diminishing, ameliorating or maintaining the existing disease or one or more symptoms of disease). The “therapeutically effective amount” may vary depending on the RNAi agent, how the agent is administered, the disease and its severity and the history, age, weight, family history, genetic makeup, the types of preceding or concomitant treatments, if any, and other individual characteristics of the subject to be treated.

“Prophylactically effective amount,” as used herein, is intended to include the amount of an iRNA that, when administered to a subject having a glycogen storage disease (GSD), e.g., type Ia GSD, is sufficient to prevent or ameliorate the disease or one or more symptoms of the disease. Ameliorating the disease includes slowing the course of the disease or reducing the severity of later-developing disease. The “prophylactically effective amount” may vary depending on the iRNA, how the agent is administered, the degree of risk of disease, and the history, age, weight, family history, genetic makeup, the types of preceding or concomitant treatments, if any, and other individual characteristics of the patient to be treated.

A “therapeutically-effective amount” or “prophylacticaly effective amount” also includes an amount of an RNAi agent that produces some desired local or systemic effect at a reasonable benefit/risk ratio applicable to any treatment. iRNA employed in the methods of the present invention may be administered in a sufficient amount to produce a reasonable benefit/risk ratio applicable to such treatment.

The phrase “pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human subjects and animal subjects without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

The phrase “pharmaceutically-acceptable carrier” as used herein means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, talc magnesium, calcium or zinc stearate, or steric acid), or solvent encapsulating material, involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the subject being treated. Some examples of materials which can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricating agents, such as magnesium state, sodium lauryl sulfate and talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21) polyesters, polycarbonates and/or polyanhydrides; (22) bulking agents, such as polypeptides and amino acids (23) serum component, such as serum albumin, HDL and LDL; and (22) other non-toxic compatible substances employed in pharmaceutical formulations.

The term “sample,” as used herein, includes a collection of similar fluids, cells, or tissues isolated from a subject, as well as fluids, cells, or tissues present within a subject. Examples of biological fluids include blood, serum and serosal fluids, plasma, cerebrospinal fluid, ocular fluids, lymph, urine, saliva, and the like. Tissue samples may include samples from tissues, organs or localized regions. For example, samples may be derived from particular organs, parts of organs, or fluids or cells within those organs. In certain embodiments, samples may be derived from the liver (e.g., whole liver or certain segments of liver or certain types of cells in the liver, such as, e.g., hepatocytes). In some embodiments, a “sample derived from a subject” refers to blood drawn from the subject or plasma isolated therefrom, saliva, or urine, typically a 24 hour urine sample.

II. iRNAs of the Invention

Described herein are iRNAs which inhibit the expression of a GCK gene. In one embodiment, the iRNA agent includes double stranded ribonucleic acid (dsRNA) molecules for inhibiting the expression of a GCK gene in a cell, such as a cell within a subject, e.g., a mammal, such as a human having a glycogen storage disease (GSD), e.g., type Ia GSD. The dsRNA includes an antisense strand having a region of complementarity which is complementary to at least a part of an mRNA formed in the expression of a GCK gene. The region of complementarity is about 30 nucleotides or less in length (e.g., about 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, or 18 nucleotides or less in length). Upon contact with a cell expressing the GCK gene, the iRNA inhibits the expression of the GCK gene (e.g., a human, a primate, a non-primate, or a bird GCK gene) by at least about 10% as assayed by, for example, a PCR or branched DNA (bDNA)-based method, or by a protein-based method, such as by immunofluorescence analysis, using, for example, western blotting, or flowcytometric techniques. In preferred embodiments, inhibition of expression is determined by the qPCR method provided in the examples. For in vitro assessment of activity, percent inhibition is determined using the methods provided herein at a single dose at, for example, a 10 nM duplex final concentration. For in vivo studies, the level after treatment can be compared to, for example, an appropriate historical control or a pooled population sample control to determine the level of reduction, e.g., when a baseline value is no available for the subject.

A dsRNA includes two RNA strands that are complementary and hybridize to form a duplex structure under conditions in which the dsRNA will be used. One strand of a dsRNA (the antisense strand) includes a region of complementarity that is substantially complementary, and generally fully complementary, to a target sequence. The target sequence can be derived from the sequence of an mRNA formed during the expression of a GCK gene. The other strand (the sense strand) includes a region that is complementary to the antisense strand, such that the two strands hybridize and form a duplex structure when combined under suitable conditions. As described elsewhere herein and as known in the art, the complementary sequences of a dsRNA can also be contained as self-complementary regions of a single nucleic acid molecule, as opposed to being on separate oligonucleotides.

Generally, the duplex structure is between 15 and 30 base pairs in length, e.g., between, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs in length. Ranges and lengths intermediate to the above recited ranges and lengths are also contemplated to be part of the invention.

Similarly, the region of complementarity to the target sequence is between 15 and 30 nucleotides in length, e.g., between 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides in length. Ranges and lengths intermediate to the above recited ranges and lengths are also contemplated to be part of the invention.

In some embodiments, the dsRNA is between about 15 and about 23 nucleotides in length, or between about 25 and about 30 nucleotides in length. In general, the dsRNA is long enough to serve as a substrate for the Dicer enzyme. For example, it is well known in the art that dsRNAs longer than about 21-23 nucleotides can serve as substrates for Dicer. As the ordinarily skilled person will also recognize, the region of an RNA targeted for cleavage will most often be part of a larger RNA molecule, often an mRNA molecule. Where relevant, a “part” of an mRNA target is a contiguous sequence of an mRNA target of sufficient length to allow it to be a substrate for RNAi-directed cleavage (i.e., cleavage through a RISC pathway).

One of skill in the art will also recognize that the duplex region is a primary functional portion of a dsRNA, e.g., a duplex region of about 9 to 36 base pairs, e.g., about 10-36, 11-36, 12-36, 13-36, 14-36, 15-36, 9-35, 10-35, 11-35, 12-35, 13-35, 14-35, 15-35, 9-34, 10-34, 11-34, 12-34, 13-34, 14-34, 15-34, 9-33, 10-33, 11-33, 12-33, 13-33, 14-33, 15-33, 9-32, 10-32, 11-32, 12-32, 13-32, 14-32, 15-32, 9-31, 10-31, 11-31, 12-31, 13-32, 14-31, 15-31, 15-30, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs. Thus, in one embodiment, to the extent that it becomes processed to a functional duplex, of e.g., 15-30 base pairs, that targets a desired RNA for cleavage, an RNA molecule or complex of RNA molecules having a duplex region greater than 30 base pairs is a dsRNA. Thus, an ordinarily skilled artisan will recognize that in one embodiment, a miRNA is a dsRNA. In another embodiment, a dsRNA is not a naturally occurring miRNA. In another embodiment, an iRNA agent useful to target GCK expression is not generated in the target cell by cleavage of a larger dsRNA.

A dsRNA as described herein can further include one or more single-stranded nucleotide overhangs e.g., 1, 2, 3, or 4 nucleotides. dsRNAs having at least one nucleotide overhang can have unexpectedly superior inhibitory properties relative to their blunt-ended counterparts. A nucleotide overhang can comprise or consist of a nucleotide/nucleoside analog, including a deoxynucleotide/nucleoside. The overhang(s) can be on the sense strand, the antisense strand or any combination thereof. Furthermore, the nucleotide(s) of an overhang can be present on the 5′-end, 3′-end, or both ends of either an antisense or sense strand of a dsRNA. In certain embodiments, longer, extended overhangs are possible.

A dsRNA can be synthesized by standard methods known in the art as further discussed below, e.g., by use of an automated DNA synthesizer, such as are commercially available from, for example, Biosearch™, Applied Biosystems™, Inc.

iRNA compounds of the invention may be prepared using a two-step procedure. First, the individual strands of the double stranded RNA molecule are prepared separately. Then, the component strands are annealed. The individual strands of the siRNA compound can be prepared using solution-phase or solid-phase organic synthesis or both. Organic synthesis offers the advantage that the oligonucleotide strands comprising unnatural or modified nucleotides can be easily prepared. Single-stranded oligonucleotides of the invention can be prepared using solution-phase or solid-phase organic synthesis or both.

In certain aspects, a dsRNA of the invention includes at least two nucleotide sequences, a sense sequence and an anti-sense sequence. The sense strand sequence is selected from the group of sequences provided in any of Tables 2, 3, 6, and 7 and the corresponding nucleotide sequence of the antisense strand of the sense strand is selected from the group of sequences provided in any of Tables 2, 3, 6, and 7. In this aspect, one of the two sequences is complementary to the other of the two sequences, with one of the sequences being substantially complementary to a sequence of an mRNA generated in the expression of a GCK gene. As such, in this aspect, a dsRNA will include two oligonucleotides, where one oligonucleotide is described as the sense strand in any of Tables 2, 3, 6, and 7, and the second oligonucleotide is described as the corresponding antisense strand of the sense strand in any of Tables 2, 3, 6, and 7. In one embodiment, the substantially complementary sequences of the dsRNA are contained on separate oligonucleotides. In another embodiment, the substantially complementary sequences of the dsRNA are contained on a single oligonucleotide.

It will be understood that, although the sequences in Tables 3 and 7 are described as modified and/or conjugated sequences, the RNA of the iRNA of the invention e.g., a dsRNA of the invention, may comprise any one of the sequences set forth in any one of Tables 3 and 7 that is un-modified, un-conjugated, and/or modified and/or conjugated differently than described therein.

In another aspect, a double stranded ribonucleic acid (dsRNA) of the invention for inhibiting expression of GCK comprises, consists essentially of, or consists of a sense strand and an antisense strand, wherein the sense strand comprises the nucleotide sequence of a sense strand in any of Tables 2, 3, 6, and 7 and the antisense strand comprises the nucleotide sequence of the corresponding antisense strand in any of Tables 2, 3, 6, and 7.

The skilled person is well aware that dsRNAs having a duplex structure of between about 20 and 23 base pairs, e.g., 21, base pairs have been hailed as particularly effective in inducing RNA interference (Elbashir et al., (2001) EMBO J., 20:6877-6888). However, others have found that shorter or longer RNA duplex structures can also be effective (Chu and Rana (2007) RNA 14:1714-1719; Kim et al. (2005) Nat Biotech 23:222-226). In the embodiments described above, by virtue of the nature of the oligonucleotide sequences provided herein, dsRNAs described herein can include at least one strand of a length of minimally 21 nucleotides. It can be reasonably expected that shorter duplexes minus only a few nucleotides on one or both ends can be similarly effective as compared to the dsRNAs described above. Hence, dsRNAs having a sequence of at least 15, 16, 17, 18, 19, 20, or more contiguous nucleotides derived from one of the sequences provided herein, and differing in their ability to inhibit the expression of a GCK gene by not more than about 5, 10, 15, 20, 25, or 30% inhibition from a dsRNA comprising the full sequence, are contemplated to be within the scope of the present invention.

In addition, the RNAs described herein identify a site(s) in a GCK transcript that is susceptible to RISC-mediated cleavage. As such, the present invention further features iRNAs that target within this site(s). As used herein, an iRNA is said to target within a particular site of an RNA transcript if the iRNA promotes cleavage of the transcript anywhere within that particular site. Such an iRNA will generally include at least about 15 contiguous nucleotides from one of the sequences provided herein coupled to additional nucleotide sequences taken from the region contiguous to the selected sequence in a GCK gene.

While a target sequence is generally about 15-30 nucleotides in length, there is wide variation in the suitability of particular sequences in this range for directing cleavage of any given target RNA. Various software packages and the guidelines set out herein provide guidance for the identification of optimal target sequences for any given gene target, but an empirical approach can also be taken in which a “window” or “mask” of a given size (as a non-limiting example, 21 nucleotides) is literally or figuratively (including, e.g., in silico) placed on the target RNA sequence to identify sequences in the size range that can serve as target sequences. By moving the sequence “window” progressively one nucleotide upstream or downstream of an initial target sequence location, the next potential target sequence can be identified, until the complete set of possible sequences is identified for any given target size selected. This process, coupled with systematic synthesis and testing of the identified sequences (using assays as described herein or as known in the art) to identify those sequences that perform optimally can identify those RNA sequences that, when targeted with an iRNA agent, mediate the best inhibition of target gene expression. Thus, while the sequences identified herein represent effective target sequences, it is contemplated that further optimization of inhibition efficiency can be achieved by progressively “walking the window” one nucleotide upstream or downstream of the given sequences to identify sequences with equal or better inhibition characteristics.

Further, it is contemplated that for any sequence identified herein, further optimization could be achieved by systematically either adding or removing nucleotides to generate longer or shorter sequences and testing those sequences generated by walking a window of the longer or shorter size up or down the target RNA from that point. Again, coupling this approach to generating new candidate targets with testing for effectiveness of iRNAs based on those target sequences in an inhibition assay as known in the art and/or as described herein can lead to further improvements in the efficiency of inhibition. Further still, such optimized sequences can be adjusted by, e.g., the introduction of modified nucleotides as described herein or as known in the art, addition or changes in overhang, or other modifications as known in the art and/or discussed herein to further optimize the molecule (e.g., increasing serum stability or circulating half-life, increasing thermal stability, enhancing transmembrane delivery, targeting to a particular location or cell type, increasing interaction with silencing pathway enzymes, increasing release from endosomes) as an expression inhibitor.

An iRNA agent as described herein can contain one or more mismatches to the target sequence. In one embodiment, an iRNA as described herein contains no more than 3 mismatches. If the antisense strand of the iRNA contains mismatches to a target sequence, it is preferable that the area of mismatch is not located in the center of the region of complementarity. If the antisense strand of the iRNA contains mismatches to the target sequence, it is preferable that the mismatch be restricted to be within the last 5 nucleotides from either the 5′- or 3′-end of the region of complementarity. For example, for a 23 nucleotide iRNA agent the strand which is complementary to a region of a GCK gene, generally does not contain any mismatch within the central 13 nucleotides. The methods described herein or methods known in the art can be used to determine whether an iRNA containing a mismatch to a target sequence is effective in inhibiting the expression of a GCK gene. Consideration of the efficacy of iRNAs with mismatches in inhibiting expression of a GCK gene is important, especially if the particular region of complementarity in a GCK gene is known to have polymorphic sequence variation within the population.

III. Modified iRNAs of the Invention

In one embodiment, the RNA of the iRNA of the invention e.g., a dsRNA, is un-modified, and does not comprise, e.g., chemical modifications and/or conjugations known in the art and described herein. In another embodiment, the RNA of an iRNA of the invention, e.g., a dsRNA, is chemically modified to enhance stability or other beneficial characteristics. In certain embodiments of the invention, substantially all of the nucleotides of an iRNA of the invention are modified. In other embodiments of the invention, all of the nucleotides of an iRNA of the invention are modified. iRNAs of the invention in which “substantially all of the nucleotides are modified” are largely but not wholly modified and can include not more than 5, 4, 3, 2, or 1 unmodified nucleotides. The nucleic acids featured in the invention can be synthesized and/or modified by methods well established in the art, such as those described in “Current protocols in nucleic acid chemistry,” Beaucage, S. L. et al. (Edrs.), John Wiley & Sons, Inc., New York, N.Y., USA, which is hereby incorporated herein by reference. Modifications include, for example, end modifications, e.g., 5′-end modifications (phosphorylation, conjugation, inverted linkages) or 3′-end modifications (conjugation, DNA nucleotides, inverted linkages, etc.); base modifications, e.g., replacement with stabilizing bases, destabilizing bases, or bases that base pair with an expanded repertoire of partners, removal of bases (abasic nucleotides), or conjugated bases; sugar modifications (e.g., at the 2′-position or 4′-position) or replacement of the sugar; and/or backbone modifications, including modification or replacement of the phosphodiester linkages.

Specific examples of iRNA compounds useful in the embodiments described herein include, but are not limited to RNAs containing modified backbones or no natural internucleoside linkages. RNAs having modified backbones include, among others, those that do not have a phosphorus atom in the backbone. For the purposes of this specification, and as sometimes referenced in the art, modified RNAs that do not have a phosphorus atom in their internucleoside backbone can also be considered to be oligonucleosides. In some embodiments, a modified iRNA will have a phosphorus atom in its internucleoside backbone.

Modified RNA backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3′-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3′-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3′-5′ linkages, 2′-5′-linked analogs of these, and those having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. Various salts, mixed salts and free acid forms are also included.

Representative U.S. patents that teach the preparation of the above phosphorus-containing linkages include, but are not limited to, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,195; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,316; 5,550,111; 5,563,253; 5,571,799; 5,587,361; 5,625,050; 6,028,188; 6,124,445; 6,160,109; 6,169,170; 6,172,209; 6,239,265; 6,277,603; 6,326,199; 6,346,614; 6,444,423; 6,531,590; 6,534,639; 6,608,035; 6,683,167; 6,858,715; 6,867,294; 6,878,805; 7,015,315; 7,041,816; 7,273,933; 7,321,029; and U.S. Pat. No. RE39464, the entire contents of each of which are hereby incorporated herein by reference.

Modified RNA backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatoms and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside);

siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S, and CH₂ component parts.

Representative U.S. patents that teach the preparation of the above oligonucleosides include, but are not limited to, U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,64,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and, 5,677,439, the entire contents of each of which are hereby incorporated herein by reference.

In other embodiments, suitable RNA mimetics are contemplated for use in iRNAs, in which both the sugar and the internucleoside linkage, i.e., the backbone, of the nucleotide units are replaced with novel groups. The base units are maintained for hybridization with an appropriate nucleic acid target compound. One such oligomeric compound, an RNA mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of an RNA is replaced with an amide containing backbone, in particular an aminoethylglycine backbone. The nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone. Representative U.S. patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, the entire contents of each of which are hereby incorporated herein by reference. Additional PNA compounds suitable for use in the iRNAs of the invention are described in, for example, in Nielsen et al., Science, 1991, 254, 1497-1500.

Some embodiments featured in the invention include RNAs with phosphorothioate backbones and oligonucleosides with heteroatom backbones, and in particular —CH₂—NH—CH₂—, —CH₂—N(CH₃)—O—CH₂— [known as a methylene (methylimino) or MMI backbone], —CH₂—O—N(CH₃)—CH₂—, —CH₂—N(CH₃)—N(CH₃)—CH₂— and —N(CH₃)—CH₂—CH₂— [wherein the native phosphodiester backbone is represented as —O—P—O—CH₂—] of the above-referenced U.S. Pat. No. 5,489,677, and the amide backbones of the above-referenced U.S. Pat. No. 5,602,240. In some embodiments, the RNAs featured herein have morpholino backbone structures of the above-referenced U.S. Pat. No. 5,034,506.

Modified RNAs can also contain one or more substituted sugar moieties. The iRNAs, e.g., dsRNAs, featured herein can include one of the following at the 2′-position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl can be substituted or unsubstituted C₁ to C₁₀ alkyl or C₂ to C₁₀ alkenyl and alkynyl. Exemplary suitable modifications include O[(CH₂)_(n)O]_(m)CH₃, O(CH₂)._(n)OCH₃, O(CH₂)—NH₂, O(CH₂)_(n)CH₃, O(CH₂)—ONH₂, and O(CH₂)—ON[(RCH₂)_(n)CH₃)]₂, where n and m are from 1 to about 10. In other embodiments, dsRNAs include one of the following at the 2′ position: C₁ to C₁₀ lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl, Br, CN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an iRNA, or a group for improving the pharmacodynamic properties of an iRNA, and other substituents having similar properties. In some embodiments, the modification includes a 2′-methoxyethoxy (2′-O—CH₂CH₂OCH₃, also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78:486-504) i.e., an alkoxy-alkoxy group. Another exemplary modification is 2′-dimethylaminooxyethoxy, i.e., a O(CH₂)₂ON(CH₃)₂ group, also known as 2′-DMAOE, as described in examples herein below, and 2′-dimethylaminoethoxyethoxy (also known in the art as 2′-O-dimethylaminoethoxyethyl or 2′-DMAEOE), i.e., 2′-O—CH₂—O—CH₂—N(CH₂)₂. Further exemplary modifications include: 5′-Me-2′-F nucleotides, 5′-Me-2′-OMe nucleotides, 5′-Me-2′-deoxynucleotides, (both R and S isomers in these three families); 2′-alkoxyalkyl; and 2′-NMA (N-methylacetamide).

Other modifications include 2′-methoxy (2′-OCH₃), 2′-aminopropoxy (2′-OCH₂CH₂CH₂NH₂) and 2′-fluoro (2′-F). Similar modifications can also be made at other positions on the RNA of an iRNA, particularly the 3′ position of the sugar on the 3′ terminal nucleotide or in 2′-5′ linked dsRNAs and the 5′ position of 5′ terminal nucleotide. iRNAs can also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. Representative U.S. patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; and 5,700,920, certain of which are commonly owned with the instant application. The entire contents of each of the foregoing are hereby incorporated herein by reference.

An iRNA can also include nucleobase (often referred to in the art simply as “base”) modifications or substitutions. As used herein, “unmodified” or “natural” nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified nucleobases include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl anal other 8-substituted adenines and guanines, 5-halo, particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-daazaadenine and 3-deazaguanine and 3-deazaadenine. Further nucleobases include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in Modified Nucleosides in Biochemistry, Biotechnology and Medicine, Herdewijn, P. ed. Wiley-VCH, 2008; those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J. L, ed. John Wiley & Sons, 1990, these disclosed by Englisch et al., (1991) Angewandte Chemie, International Edition, 30:613, and those disclosed by Sanghvi, Y S., Chapter 15, dsRNA Research and Applications, pages 289-302, Crooke, S. T. and Lebleu, B., Ed., CRC Press, 1993. Certain of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds featured in the invention. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2° C. (Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., Eds., dsRNA Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278) and are exemplary base substitutions, even more particularly when combined with 2′-O-methoxyethyl sugar modifications.

Representative U.S. patents that teach the preparation of certain of the above noted modified nucleobases as well as other modified nucleobases include, but are not limited to, the above noted U.S. Pat. Nos. 3,687,808, 4,845,205; 5,130,30; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,614,617; 5,681,941; 5,750,692; 6,015,886; 6,147,200; 6,166,197; 6,222,025; 6,235,887; 6,380,368; 6,528,640; 6,639,062; 6,617,438; 7,045,610; 7,427,672; and 7,495,088, the entire contents of each of which are hereby incorporated herein by reference.

The RNA of an iRNA can also be modified to include one or more locked nucleic acids (LNA). A locked nucleic acid is a nucleotide having a modified ribose moiety in which the ribose moiety comprises an extra bridge connecting the 2′ and 4′ carbons. This structure effectively “locks” the ribose in the 3′-endo structural conformation. The addition of locked nucleic acids to siRNAs has been shown to increase siRNA stability in serum, and to reduce off-target effects (Elmen, J. et al., (2005) Nucleic Acids Research 33(1):439-447; Mook, O R. et al., (2007) Mol Canc Ther 6(3):833-843; Grunweller, A. et al., (2003) Nucleic Acids Research 31(12):3185-3193).

In some embodiments, the iRNA of the invention comprises one or more monomers that are UNA (unlocked nucleic acid) nucleotides. UNA is unlocked acyclic nucleic acid, wherein any of the bonds of the sugar has been removed, forming an unlocked “sugar” residue. In one example, UNA also encompasses monomer with bonds between C1′-C4′ have been removed (i.e. the covalent carbon-oxygen-carbon bond between the C1′ and C4′ carbons). In another example, the C2′-C3′ bond (i.e. the covalent carbon-carbon bond between the C2′ and C3′ carbons) of the sugar has been removed (see Nuc. Acids Symp. Series, 52, 133-134 (2008) and Fluiter et al., Mol. Biosyst., 2009, 10, 1039 hereby incorporated by reference).

Representative U.S. publications that teach the preparation of UNA include, but are not limited to, U.S. Pat. No. 8,314,227; and US Patent Publication Nos. 2013/0096289; 2013/0011922; and 2011/0313020, the entire contents of each of which are hereby incorporated herein by reference.

The RNA of an iRNA can also be modified to include one or more bicyclic sugar moities. A “bicyclic sugar” is a furanosyl ring modified by the bridging of two atoms. A “bicyclic nucleoside” (“BNA”) is a nucleoside having a sugar moiety comprising a bridge connecting two carbon atoms of the sugar ring, thereby forming a bicyclic ring system. In certain embodiments, the bridge connects the 4′-carbon and the 2′-carbon of the sugar ring. Thus, in some embodiments an agent of the invention may include one or more locked nucleic acids (LNA). A locked nucleic acid is a nucleotide having a modified ribose moiety in which the ribose moiety comprises an extra bridge connecting the 2′ and 4′ carbons. In other words, an LNA is a nucleotide comprising a bicyclic sugar moiety comprising a 4′-CH2-O-2′ bridge. This structure effectively “locks” the ribose in the 3′-endo structural conformation. The addition of locked nucleic acids to siRNAs has been shown to increase siRNA stability in serum, and to reduce off-target effects (Elmen, J. et al., (2005) Nucleic Acids Research 33(1):439-447; Mook, O R. et al., (2007) Mol Canc Ther 6(3):833-843; Grunweller, A. et al., (2003) Nucleic Acids Research 31(12):3185-3193). Examples of bicyclic nucleosides for use in the polynucleotides of the invention include without limitation nucleosides comprising a bridge between the 4′ and the 2′ ribosyl ring atoms. In certain embodiments, the antisense polynucleotide agents of the invention include one or more bicyclic nucleosides comprising a 4′ to 2′ bridge. Examples of such 4′ to 2′ bridged bicyclic nucleosides, include but are not limited to 4′-(CH2)-O-2′ (LNA); 4′-(CH2)-S-2′; 4′-(CH2)2-O-2′ (ENA); 4′-CH(CH3)-O-2′ (also referred to as “constrained ethyl” or “cEt”) and 4′-CH(CH2OCH3)-O-2′ (and analogs thereof; see, e.g., U.S. Pat. No. 7,399,845); 4′-C(CH3)(CH3)-O-2′ (and analogs thereof; see e.g., U.S. Pat. No. 8,278,283); 4′-CH2-N(OCH3)-2′ (and analogs thereof; see e.g., U.S. Pat. No. 8,278,425); 4′-CH2-O—N(CH3)-2′ (see, e.g., U.S. Patent Publication No. 2004/0171570); 4′-CH2-N(R)—O-2′, wherein R is H, C1-C12 alkyl, or a protecting group (see, e.g., U.S. Pat. No. 7,427,672); 4′-CH2-C(H)(CH3)-2′ (see, e.g., Chattopadhyaya et al., J. Org. Chem., 2009, 74, 118-134); and 4′-CH2-C(═CH2)-2′ (and analogs thereof; see, e.g., U.S. Pat. No. 8,278,426). The entire contents of each of the foregoing are hereby incorporated herein by reference.

Additional representative U.S. patents and US patent Publications that teach the preparation of locked nucleic acid nucleotides include, but are not limited to, the following: U.S. Pat. Nos. 6,268,490; 6,525,191; 6,670,461; 6,770,748; 6,794,499; 6,998,484; 7,053,207; 7,034,133; 7,084,125; 7,399,845; 7,427,672; 7,569,686; 7,741,457; 8,022,193; 8,030,467; 8,278,425; 8,278,426; 8,278,283; US 2008/0039618; and US 2009/0012281, the entire contents of each of which are hereby incorporated herein by reference.

Any of the foregoing bicyclic nucleosides can be prepared having one or more stereochemical sugar configurations including for example α-L-ribofuranose and β-D-ribofuranose (see WO 99/14226).

The RNA of an iRNA can also be modified to include one or more constrained ethyl nucleotides. As used herein, a “constrained ethyl nucleotide” or “cEt” is a locked nucleic acid comprising a bicyclic sugar moiety comprising a 4′-CH(CH3)-0-2′ bridge. In one embodiment, a constrained ethyl nucleotide is in the S conformation referred to herein as “S-cEt.”

An iRNA of the invention may also include one or more “conformationally restricted nucleotides” (“CRN”). CRN are nucleotide analogs with a linker connecting the C2′ and C4′ carbons of ribose or the C3 and —C5′ carbons of ribose. CRN lock the ribose ring into a stable conformation and increase the hybridization affinity to mRNA. The linker is of sufficient length to place the oxygen in an optimal position for stability and affinity resulting in less ribose ring puckering.

Representative publications that teach the preparation of certain of the above noted CRN include, but are not limited to, US Patent Publication No. 2013/0190383; and PCT publication WO 2013/036868, the entire contents of each of which are hereby incorporated herein by reference.

Potentially stabilizing modifications to the ends of RNA molecules can include N-(acetylaminocaproyl)-4-hydroxyprolinol (Hyp-C6-NHAc), N-(caproyl-4-hydroxyprolinol (Hyp-C6), N-(acetyl-4-hydroxyprolinol (Hyp-NHAc), thymidine-2′-O-deoxythymidine (ether), N-(aminocaproyl)-4-hydroxyprolinol (Hyp-C6-amino), 2-docosanoyl-uridine-3″-phosphate, inverted base dT(idT) and others. Disclosure of this modification can be found in PCT Publication No. WO 2011/005861.

Other modifications of the nucleotides of an iRNA of the invention include a 5′ phosphate or 5′ phosphate mimic, e.g., a 5′-terminal phosphate or phosphate mimic on the antisense strand of an RNAi agent. Suitable phosphate mimics are disclosed in, for example US Patent Publication No. 2012/0157511, the entire contents of which are incorporated herein by reference.

IV. iRNAs Conjugated to Ligands

Another modification of the RNA of an iRNA of the invention involves chemically linking to the RNA one or more ligands, moieties or conjugates that enhance the activity, cellular distribution or cellular uptake of the iRNA. Such moieties include but are not limited to lipid moieties such as a cholesterol moiety (Letsinger et al., (1989) Proc. Natl. Acid. Sci. USA, 86: 6553-6556), cholic acid (Manoharan et al., (1994) Biorg. Med. Chem. Let., 4:1053-1060), a thioether, e.g., beryl-S-tritylthiol (Manoharan et al., (1992) Ann. N.Y. Acad. Sci., 660:306-309; Manoharan et al., (1993) Biorg. Med. Chem. Let., 3:2765-2770), a thiocholesterol (Oberhauser et al., (1992) Nucl. Acids Res., 20:533-538), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., (1991) EMBO J, 10:1111-1118; Kabanov et al., (1990) FEBS Lett., 259:327-330; Svinarchuk et al., (1993) Biochimie, 75:49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethyl-ammonium 1,2-di-O-hexadecyl-rac-glycero-3-phosphonate (Manoharan et al., (1995) Tetrahedron Lett., 36:3651-3654; Shea et al., (1990) Nucl. Acids Res., 18:3777-3783), a polyamine or a polyethylene glycol chain (Manoharan et al., (1995) Nucleosides & Nucleotides, 14:969-973), or adamantane acetic acid (Manoharan et al., (1995) Tetrahedron Lett., 36:3651-3654), a palmityl moiety (Mishra et al., (1995) Biochim. Biophys. Acta,1264:229-237), or an octadecylamine or hexylamino-carbonyloxycholesterol moiety (Crooke et al., (1996) J. Pharmacol. Exp. Ther., 277:923-937).

In one embodiment, a ligand alters the distribution, targeting or lifetime of an iRNA agent into which it is incorporated. In preferred embodiments a ligand provides an enhanced affinity for a selected target, e.g., molecule, cell or cell type, compartment, e.g., a cellular or organ compartment, tissue, organ or region of the body, as, e.g., compared to a species absent such a ligand. Preferred ligands will not take part in duplex pairing in a duplexed nucleic acid.

Ligands can include a naturally occurring substance, such as a protein (e.g., human serum albumin (HSA), low-density lipoprotein (LDL), or globulin); carbohydrate (e.g., a dextran, pullulan, chitin, chitosan, inulin, cyclodextrin, N-acetylglucosamine, N-acetylgalactosamine or hyaluronic acid); or a lipid. The ligand can also be a recombinant or synthetic molecule, such as a synthetic polymer, e.g., a synthetic polyamino acid. Examples of polyamino acids include polyamino acid is a polylysine (PLL), poly L-aspartic acid, poly L-glutamic acid, styrene-maleic acid anhydride copolymer, poly(L-lactide-co-glycolied) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacryllic acid), N-isopropylacrylamide polymers, or polyphosphazine. Example of polyamines include: polyethylenimine, polylysine (PLL), spermine, spermidine, polyamine, pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer polyamine, arginine, amidine, protamine, cationic lipid, cationic porphyrin, quaternary salt of a polyamine, or an alpha helical peptide.

Ligands can also include targeting groups, e.g., a cell or tissue targeting agent, e.g., a lectin, glycoprotein, lipid or protein, e.g., an antibody, that binds to a specified cell type such as a kidney cell. A targeting group can be a thyrotropin, melanotropin, lectin, glycoprotein, surfactant protein A, mucin carbohydrate, multivalent lactose, monovalent or multivalent galactose, N-acetyl-galactosamine, N-acetyl-gulucosamine multivalent mannose, multivalent fucose, glycosylated polyaminoacids, transferrin, bisphosphonate, polyglutamate, polyaspartate, a lipid, cholesterol, a steroid, bile acid, folate, vitamin B12, vitamin A, biotin, or an RGD peptide or RGD peptide mimetic. In certain embodiments, ligands include monovalent or multivalent galactose. In certain embodiments, ligands include cholesterol.

Other examples of ligands include dyes, intercalating agents (e.g. acridines), cross-linkers (e.g. psoralen, mitomycin C), porphyrins (TPPC4, texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g. EDTA), lipophilic molecules, e.g., cholesterol, cholic acid, adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone, 1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine), and peptide conjugates (e.g., antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]₂, polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes, haptens (e.g. biotin), transport/absorption facilitators (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bisimidazole, histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+ complexes of tetraazamacrocycles), dinitrophenyl, HRP, or AP.

Ligands can be proteins, e.g., glycoproteins, or peptides, e.g., molecules having a specific affinity for a co-ligand, or antibodies e.g., an antibody, that binds to a specified cell type such as a hepatic cell. Ligands can also include hormones and hormone receptors. They can also include non-peptidic species, such as lipids, lectins, carbohydrates, vitamins, cofactors, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-gulucosamine multivalent mannose, or multivalent fucose. The ligand can be, for example, a lipopolysaccharide, an activator of p38 MAP kinase, or an activator of NF-κB.

The ligand can be a substance, e.g., a drug, which can increase the uptake of the iRNA agent into the cell, for example, by disrupting the cell's cytoskeleton, e.g., by disrupting the cell's microtubules, microfilaments, and/or intermediate filaments. The drug can be, for example, taxon, vincristine, vinblastine, cytochalasin, nocodazole, japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, or myoservin.

In some embodiments, a ligand attached to an iRNA as described herein acts as a pharmacokinetic modulator (PK modulator). PK modulators include lipophiles, bile acids, steroids, phospholipid analogues, peptides, protein binding agents, PEG, vitamins etc. Exemplary PK modulators include, but are not limited to, cholesterol, fatty acids, cholic acid, lithocholic acid, dialkylglycerides, diacylglyceride, phospholipids, sphingolipids, naproxen, ibuprofen, vitamin E, biotin etc. Oligonucleotides that comprise a number of phosphorothioate linkages are also known to bind to serum protein, thus short oligonucleotides, e.g., oligonucleotides of about 5 bases, 10 bases, 15 bases or 20 bases, comprising multiple of phosphorothioate linkages in the backbone are also amenable to the present invention as ligands (e.g. as PK modulating ligands). In addition, aptamers that bind serum components (e.g. serum proteins) are also suitable for use as PK modulating ligands in the embodiments described herein.

Ligand-conjugated oligonucleotides of the invention may be synthesized by the use of an oligonucleotide that bears a pendant reactive functionality, such as that derived from the attachment of a linking molecule onto the oligonucleotide (described below). This reactive oligonucleotide may be reacted directly with commercially-available ligands, ligands that are synthesized bearing any of a variety of protecting groups, or ligands that have a linking moiety attached thereto.

The oligonucleotides used in the conjugates of the present invention may be conveniently and routinely made through the well-known technique of solid-phase synthesis. Equipment for such synthesis is sold by several vendors including, for example, Applied Biosystems™ (Foster City, Calif.). Any other means for such synthesis known in the art may additionally or alternatively be employed. It is also known to use similar techniques to prepare other oligonucleotides, such as the phosphorothioates and alkylated derivatives.

In the ligand-conjugated oligonucleotides and ligand-molecule bearing sequence-specific linked nucleosides of the present invention, the oligonucleotides and oligonucleosides may be assembled on a suitable DNA synthesizer utilizing standard nucleotide or nucleoside precursors, or nucleotide or nucleoside conjugate precursors that already bear the linking moiety, ligand-nucleotide or nucleoside-conjugate precursors that already bear the ligand molecule, or non-nucleoside ligand-bearing building blocks.

When using nucleotide-conjugate precursors that already bear a linking moiety, the synthesis of the sequence-specific linked nucleosides is typically completed, and the ligand molecule is then reacted with the linking moiety to form the ligand-conjugated oligonucleotide. In some embodiments, the oligonucleotides or linked nucleosides of the present invention are synthesized by an automated synthesizer using phosphoramidites derived from ligand-nucleoside conjugates in addition to the standard phosphoramidites and non-standard phosphoramidites that are commercially available and routinely used in oligonucleotide synthesis.

A. Lipid Conjugates

In one embodiment, the ligand or conjugate is a lipid or lipid-based molecule. Such a lipid or lipid-based molecule preferably binds a serum protein, e.g., human serum albumin (HSA). An HSA binding ligand allows for distribution of the conjugate to a target tissue, e.g., a non-kidney target tissue of the body. For example, the target tissue can be the liver, including parenchymal cells of the liver. Other molecules that can bind HSA can also be used as ligands. For example, neproxin or aspirin can be used. A lipid or lipid-based ligand can (a) increase resistance to degradation of the conjugate, (b) increase targeting or transport into a target cell or cell membrane, and/or (c) can be used to adjust binding to a serum protein, e.g., HSA.

A lipid based ligand can be used to inhibit, e.g., control the binding of the conjugate to a target tissue. For example, a lipid or lipid-based ligand that binds to HSA more strongly will be less likely to be targeted to the kidney and therefore less likely to be cleared from the body. A lipid or lipid-based ligand that binds to HSA less strongly can be used to target the conjugate to the kidney.

In a preferred embodiment, the lipid based ligand binds HSA. Preferably, it binds HSA with a sufficient affinity such that the conjugate will be preferably distributed to a non-kidney tissue. However, it is preferred that the affinity not be so strong that the HSA-ligand binding cannot be reversed.

In another preferred embodiment, the lipid based ligand binds HSA weakly or not at all, such that the conjugate will be preferably distributed to the kidney. Other moieties that target to kidney cells can also be used in place of or in addition to the lipid based ligand.

In another aspect, the ligand is a moiety, e.g., a vitamin, which is taken up by a target cell, e.g., a proliferating cell. These are particularly useful for treating disorders characterized by unwanted cell proliferation, e.g., of the malignant or non-malignant type, e.g., cancer cells. Exemplary vitamins include vitamin A, E, and K. Other exemplary vitamins include are B vitamin, e.g., folic acid, B12, riboflavin, biotin, pyridoxal or other vitamins or nutrients taken up by target cells such as liver cells. Also included are HSA and low density lipoprotein (LDL).

B. Cell Permeation Agents

In another aspect, the ligand is a cell-permeation agent, preferably a helical cell-permeation agent. Preferably, the agent is amphipathic. An exemplary agent is a peptide such as tat or antennopedia. If the agent is a peptide, it can be modified, including a peptidylmimetic, invertomers, non-peptide or pseudo-peptide linkages, and use of D-amino acids. The helical agent is preferably an alpha-helical agent, which preferably has a lipophilic and a lipophobic phase.

The ligand can be a peptide or peptidomimetic. A peptidomimetic (also referred to herein as an oligopeptidomimetic) is a molecule capable of folding into a defined three-dimensional structure similar to a natural peptide. The attachment of peptide and peptidomimetics to iRNA agents can affect pharmacokinetic distribution of the iRNA, such as by enhancing cellular recognition and absorption. The peptide or peptidomimetic moiety can be about 5-50 amino acids long, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long.

A peptide or peptidomimetic can be, for example, a cell permeation peptide, cationic peptide, amphipathic peptide, or hydrophobic peptide (e.g., consisting primarily of Tyr, Trp or Phe). The peptide moiety can be a dendrimer peptide, constrained peptide or crosslinked peptide. In another alternative, the peptide moiety can include a hydrophobic membrane translocation sequence (MTS). An exemplary hydrophobic MTS-containing peptide is RFGF having the amino acid sequence AAVALLPAVLLALLAP (SEQ ID NO: 25). An RFGF analogue (e.g., amino acid sequence AALLPVLLAAP (SEQ ID NO: 26) containing a hydrophobic MTS can also be a targeting moiety. The peptide moiety can be a “delivery” peptide, which can carry large polar molecules including peptides, oligonucleotides, and protein across cell membranes. For example, sequences from the HIV Tat protein (GRKKRRQRRRPPQ (SEQ ID NO: 27) and the Drosophila Antennapedia protein (RQIKIWFQNRRMKWKK (SEQ ID NO: 28) have been found to be capable of functioning as delivery peptides. A peptide or peptidomimetic can be encoded by a random sequence of DNA, such as a peptide identified from a phage-display library, or one-bead-one-compound (OBOC) combinatorial library (Lam et al., Nature, 354:82-84, 1991). Examples of a peptide or peptidomimetic tethered to a dsRNA agent via an incorporated monomer unit for cell targeting purposes is an arginine-glycine-aspartic acid (RGD)-peptide, or RGD mimic A peptide moiety can range in length from about 5 amino acids to about 40 amino acids. The peptide moieties can have a structural modification, such as to increase stability or direct conformational properties. Any of the structural modifications described below can be utilized.

An RGD peptide for use in the compositions and methods of the invention may be linear or cyclic, and may be modified, e.g., glyciosylated or methylated, to facilitate targeting to a specific tissue(s). RGD-containing peptides and peptidiomimemtics may include D-amino acids, as well as synthetic RGD mimics. In addition to RGD, one can use other moieties that target the integrin ligand. Preferred conjugates of this ligand target PECAM-1 or VEGF.

A “cell permeation peptide” is capable of permeating a cell, e.g., a microbial cell, such as a bacterial or fungal cell, or a mammalian cell, such as a human cell. A microbial cell-permeating peptide can be, for example, a α-helical linear peptide (e.g., LL-37 or Ceropin P1), a disulfide bond-containing peptide (e.g., α-defensin, β-defensin or bactenecin), or a peptide containing only one or two dominating amino acids (e.g., PR-39 or indolicidin). A cell permeation peptide can also include a nuclear localization signal (NLS). For example, a cell permeation peptide can be a bipartite amphipathic peptide, such as MPG, which is derived from the fusion peptide domain of HIV-1 gp41 and the NLS of SV40 large T antigen (Simeoni et al., Nucl. Acids Res. 31:2717-2724, 2003).

C. Carbohydrate Conjugates

In some embodiments of the compositions and methods of the invention, an iRNA oligonucleotide further comprises a carbohydrate. The carbohydrate conjugated iRNA are advantageous for the in vivo delivery of nucleic acids, as well as compositions suitable for in vivo therapeutic use, as described herein. As used herein, “carbohydrate” refers to a compound which is either a carbohydrate per se made up of one or more monosaccharide units having at least 6 carbon atoms (which can be linear, branched or cyclic) with an oxygen, nitrogen, or sulfur atom bonded to each carbon atom; or a compound having as a part thereof a carbohydrate moiety made up of one or more monosaccharide units each having at least six carbon atoms (which can be linear, branched or cyclic), with an oxygen, nitrogen, or sulfur atom bonded to each carbon atom. Representative carbohydrates include the sugars (mono-, di-, tri- and oligosaccharides containing from about 4, 5, 6, 7, 8, or 9 monosaccharide units), and polysaccharides such as starches, glycogen, cellulose and polysaccharide gums. Specific monosaccharides include C5 and above (e.g., C5, C6, C7, or C8) sugars; di- and trisaccharides include sugars having two or three monosaccharide units (e.g., C5, C6, C7, or C8).

In one embodiment, a carbohydrate conjugate for use in the compositions and methods of the invention is a monosaccharide.

In one embodiment, a carbohydrate conjugate for use in the compositions and methods of the invention is selected from the group consisting of:

In one embodiment, the monosaccharide is an N-acetylgalactosamine, such as

Another representative carbohydrate conjugate for use in the embodiments described herein includes, but is not limited to,

when one of X or Y is an oligonucleotide, the other is a hydrogen.

In certain embodiments of the invention, the GalNAc or GalNAc derivative is attached to an iRNA agent of the invention via a monovalent linker. In some embodiments, the GalNAc or GalNAc derivative is attached to an iRNA agent of the invention via a bivalent linker. In yet other embodiments of the invention, the GalNAc or GalNAc derivative is attached to an iRNA agent of the invention via a trivalent linker.

In one embodiment, the double stranded RNAi agents of the invention comprise one GalNAc or GalNAc derivative attached to the iRNA agent. In another embodiment, the double stranded RNAi agents of the invention comprise a plurality (e.g., 2, 3, 4, 5, or 6) GalNAc or GalNAc derivatives, each independently attached to a plurality of nucleotides of the double stranded RNAi agent through a plurality of monovalent linkers.

In some embodiments, for example, when the two strands of an iRNA agent of the invention are part of one larger molecule connected by an uninterrupted chain of nucleotides between the 3′-end of one strand and the 5′-end of the respective other strand forming a hairpin loop comprising, a plurality of unpaired nucleotides, each unpaired nucleotide within the hairpin loop may independently comprise a GalNAc or GalNAc derivative attached via a monovalent linker. The hairpin loop may also be formed by an extended overhang in one strand of the duplex.

In some embodiments, the carbohydrate conjugate further comprises one or more additional ligands as described above, such as, but not limited to, a PK modulator and/or a cell permeation peptide.

Additional carbohydrate conjugates (and linkers) suitable for use in the present invention include those described in PCT Publication Nos. WO 2014/179620 and WO 2014/179627, the entire contents of each of which are incorporated herein by reference.

D. Linkers

In some embodiments, the conjugate or ligand described herein can be attached to an iRNA oligonucleotide with various linkers that can be cleavable or non cleavable.

The term “linker” or “linking group” means an organic moiety that connects two parts of a compound, e.g., covalently attaches two parts of a compound. Linkers typically comprise a direct bond or an atom such as oxygen or sulfur, a unit such as NR8, C(O), C(O)NH, SO, SO₂, SO₂NH or a chain of atoms, such as, but not limited to, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl, heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, alkylarylalkyl, alkylarylalkenyl, alkylarylalkynyl, alkenylarylalkyl, alkenylarylalkenyl, alkenylarylalkynyl, alkynylarylalkyl, alkynylarylalkenyl, alkynylarylalkynyl, alkylheteroarylalkyl, alkylheteroarylalkenyl, alkylheteroarylalkynyl, alkenylheteroarylalkyl, alkenylheteroarylalkenyl, alkenylheteroarylalkynyl, alkynylheteroarylalkyl, alkynylheteroarylalkenyl, alkynylheteroarylalkynyl, alkylheterocyclylalkyl, alkylheterocyclylalkenyl, alkylhererocyclylalkynyl, alkenylheterocyclylalkyl, alkenylheterocyclylalkenyl, alkenylheterocyclylalkynyl, alkynylheterocyclylalkyl, alkynylheterocyclylalkenyl, alkynylheterocyclylalkynyl, alkylaryl, alkenylaryl, alkynylaryl, alkylheteroaryl, alkenylheteroaryl, alkynylhereroaryl, which one or more methylenes can be interrupted or terminated by O, S, S(O), SO₂, NR8), C(O), substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclic; where R8 is hydrogen, acyl, aliphatic or substituted aliphatic. In one embodiment, the linker is between about 1-24 atoms, 2-24, 3-24, 4-24, 5-24, 6-24, 6-18, 7-18, 8-18 atoms, 7-17, 8-17, 6-16, 7-17, or 8-16 atoms.

A cleavable linking group is one which is sufficiently stable outside the cell, but which upon entry into a target cell is cleaved to release the two parts the linker is holding together. In a preferred embodiment, the cleavable linking group is cleaved at least about 10 times, 20, times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times or more, or at least about 100 times faster in a target cell or under a first reference condition (which can, e.g., be selected to mimic or represent intracellular conditions) than in the blood of a subject, or under a second reference condition (which can, e.g., be selected to mimic or represent conditions found in the blood or serum).

Cleavable linking groups are susceptible to cleavage agents, e.g., pH, redox potential or the presence of degradative molecules. Generally, cleavage agents are more prevalent or found at higher levels or activities inside cells than in serum or blood. Examples of such degradative agents include: redox agents which are selected for particular substrates or which have no substrate specificity, including, e.g., oxidative or reductive enzymes or reductive agents such as mercaptans, present in cells, that can degrade a redox cleavable linking group by reduction; esterases; endosomes or agents that can create an acidic environment, e.g., those that result in a pH of five or lower; enzymes that can hydrolyze or degrade an acid cleavable linking group by acting as a general acid, peptidases (which can be substrate specific), and phosphatases.

A cleavable linkage group, such as a disulfide bond can be susceptible to pH. The pH of human serum is 7.4, while the average intracellular pH is slightly lower, ranging from about 7.1-7.3. Endosomes have a more acidic pH, in the range of 5.5-6.0, and lysosomes have an even more acidic pH at around 5.0. Some linkers will have a cleavable linking group that is cleaved at a preferred pH, thereby releasing a cationic lipid from the ligand inside the cell, or into the desired compartment of the cell.

A linker can include a cleavable linking group that is cleavable by a particular enzyme. The type of cleavable linking group incorporated into a linker can depend on the cell to be targeted. For example, a liver-targeting ligand can be linked to a cationic lipid through a linker that includes an ester group. Liver cells are rich in esterases, and therefore the linker will be cleaved more efficiently in liver cells than in cell types that are not esterase-rich. Other cell-types rich in esterases include cells of the lung, renal cortex, and testis.

Linkers that contain peptide bonds can be used when targeting cell types rich in peptidases, such as liver cells and synoviocytes.

In general, the suitability of a candidate cleavable linking group can be evaluated by testing the ability of a degradative agent (or condition) to cleave the candidate linking group. It will also be desirable to also test the candidate cleavable linking group for the ability to resist cleavage in the blood or when in contact with other non-target tissue. Thus, one can determine the relative susceptibility to cleavage between a first and a second condition, where the first is selected to be indicative of cleavage in a target cell and the second is selected to be indicative of cleavage in other tissues or biological fluids, e.g., blood or serum. The evaluations can be carried out in cell free systems, in cells, in cell culture, in organ or tissue culture, or in whole animals. It can be useful to make initial evaluations in cell-free or culture conditions and to confirm by further evaluations in whole animals. In preferred embodiments, useful candidate compounds are cleaved at least about 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, or about 100 times faster in the cell (or under in vitro conditions selected to mimic intracellular conditions) as compared to blood or serum (or under in vitro conditions selected to mimic extracellular conditions).

i. Redox Cleavable Linking Groups

In one embodiment, a cleavable linking group is a redox cleavable linking group that is cleaved upon reduction or oxidation. An example of reductively cleavable linking group is a disulphide linking group (—S—S—). To determine if a candidate cleavable linking group is a suitable “reductively cleavable linking group,” or for example is suitable for use with a particular iRNA moiety and particular targeting agent one can look to methods described herein. For example, a candidate can be evaluated by incubation with dithiothreitol (DTT), or other reducing agent using reagents know in the art, which mimic the rate of cleavage which would be observed in a cell, e.g., a target cell. The candidates can also be evaluated under conditions which are selected to mimic blood or serum conditions. In one, candidate compounds are cleaved by at most about 10% in the blood. In other embodiments, useful candidate compounds are degraded at least about 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, or about 100 times faster in the cell (or under in vitro conditions selected to mimic intracellular conditions) as compared to blood (or under in vitro conditions selected to mimic extracellular conditions). The rate of cleavage of candidate compounds can be determined using standard enzyme kinetics assays under conditions chosen to mimic intracellular media and compared to conditions chosen to mimic extracellular media.

ii. Phosphate-Based Cleavable Linking Groups

In another embodiment, a cleavable linker comprises a phosphate-based cleavable linking group. A phosphate-based cleavable linking group is cleaved by agents that degrade or hydrolyze the phosphate group. An example of an agent that cleaves phosphate groups in cells are enzymes such as phosphatases in cells. Examples of phosphate-based linking groups are —O—P(O)(ORk)-O—, —O—P(S)(ORk)-O—, —O—P(S)(SRk)-O—, —S—P(O)(ORk)-O—, —O—P(O)(ORk)-S—, —S—P(O)(ORk)-S—, —O—P(S)(ORk)-S—, —S—P(S)(ORk)-O—, —O—P(O)(Rk)-O—, —O—P(S)(Rk)-O—, —S—P(O)(Rk)-O—, —S—P(S)(Rk)-O—, —S—P(O)(Rk)-S—, —O—P(S)(Rk)-S—. Preferred embodiments are —O—P(O)(OH)—O—, —O—P(S)(OH)—O—, —O—P(S)(SH)—O—, —S—P(O)(OH)—O—, —O—P(O)(OH)—S—, —S—P(O)(OH)—S—, —O—P(S)(OH)—S—, —S—P(S)(OH)—O—, —O—P(O)(H)—O—, —O—P(S)(H)—O—, —S—P(O)(H)—O—, —S—P(S)(H)—O—, —S—P(O)(H)—S—, —O—P(S)(H)—S—. A preferred embodiment is —O—P(O)(OH)—O—. These candidates can be evaluated using methods analogous to those described above.

iii. Acid Cleavable Linking Groups

In another embodiment, a cleavable linker comprises an acid cleavable linking group. An acid cleavable linking group is a linking group that is cleaved under acidic conditions. In preferred embodiments acid cleavable linking groups are cleaved in an acidic environment with a pH of about 6.5 or lower (e.g., about 6.0, 5.75, 5.5, 5.25, 5.0, or lower), or by agents such as enzymes that can act as a general acid. In a cell, specific low pH organelles, such as endosomes and lysosomes can provide a cleaving environment for acid cleavable linking groups. Examples of acid cleavable linking groups include but are not limited to hydrazones, esters, and esters of amino acids. Acid cleavable groups can have the general formula —C═NN—, C(O)O, or —OC(O). A preferred embodiment is when the carbon attached to the oxygen of the ester (the alkoxy group) is an aryl group, substituted alkyl group, or tertiary alkyl group such as dimethyl pentyl or t-butyl. These candidates can be evaluated using methods analogous to those described above.

iv. Ester-Based Linking Groups

In another embodiment, a cleavable linker comprises an ester-based cleavable linking group. An ester-based cleavable linking group is cleaved by enzymes such as esterases and amidases in cells. Examples of ester-based cleavable linking groups include but are not limited to esters of alkylene, alkenylene, and alkynylene groups. Ester cleavable linking groups have the general formula —C(O)O—, or —OC(O)—. These candidates can be evaluated using methods analogous to those described above.

v. Peptide-Based Cleaving Groups

In yet another embodiment, a cleavable linker comprises a peptide-based cleavable linking group. A peptide-based cleavable linking group is cleaved by enzymes such as peptidases and proteases in cells. Peptide-based cleavable linking groups are peptide bonds formed between amino acids to yield oligopeptides (e.g., dipeptides, tripeptides, etc.) and polypeptides. Peptide-based cleavable groups do not include the amide group (—C(O)NH—). The amide group can be formed between any alkylene, alkenylene, or alkynelene. A peptide bond is a special type of amide bond formed between amino acids to yield peptides and proteins. The peptide based cleavage group is generally limited to the peptide bond (i.e., the amide bond) formed between amino acids yielding peptides and proteins and does not include the entire amide functional group. Peptide-based cleavable linking groups have the general formula —NHCHRAC(O)NHCHRBC(O)—, where RA and RB are the R groups of the two adjacent amino acids. These candidates can be evaluated using methods analogous to those described above.

In one embodiment, an iRNA of the invention is conjugated to a carbohydrate through a linker. Non-limiting examples of iRNA carbohydrate conjugates with linkers of the compositions and methods of the invention include, but are not limited to,

when one of X or Y is an oligonucleotide, the other is a hydrogen.

In certain embodiments of the compositions and methods of the invention, a ligand is one or more GalNAc (N-acetylgalactosamine) derivatives attached through a bivalent or trivalent branched linker.

In one embodiment, a dsRNA of the invention is conjugated to a bivalent or trivalent branched linker selected from the group of structures shown in any of formula (XXXII)-(XXXV):

wherein: q2A, q2B, q3A, q3B, q4A, q4B, q5A, q5B and q5C represent independently for each occurrence 0-20 and wherein the repeating unit can be the same or different; P^(2A), P^(2B), P^(3A), P^(3B), P^(4A), P^(4B), P^(5A), P^(5B), P^(5C), T^(2A), T^(2B), T^(3A), T^(3B), T^(4A), T^(4B), T^(4A), T^(5B), T^(5C) are each independently for each occurrence absent, CO, NH, O, S, OC(O), NHC(O), CH₂, CH₂NH or CH₂O;

Q^(2A), Q^(2B), Q^(3A), Q^(3B), Q^(4A), Q^(4B), Q^(5A), Q^(5B), Q^(5C) are independently for each occurrence absent, alkylene, substituted alkylene wherein one or more methylenes can be interrupted or terminated by one or more of O, S, S(O), SO₂, N(R^(N)), C(R′)═C(R″), C≡C or C(O); R^(2A), R^(2B), R^(3A), R^(3B), R^(4A), R^(4B), R^(5A), R^(5B), R^(5C) are each independently for each occurrence absent, NH, O, S, CH₂, C(O)O, C(O)NH, NHCH(R^(a))C(O), —C(O)—CH(R^(a))—NH—, CO,

or heterocyclyl;

L^(2A), L^(2B), L^(3A), L^(3B), L^(4A), L^(4B), L^(5A), L^(5B) and L^(5C) represent the ligand; i.e. each independently for each occurrence a monosaccharide (such as GalNAc), disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, or polysaccharide; and R^(a) is H or amino acid side chain. Trivalent conjugating GalNAc derivatives are particularly useful for use with RNAi agents for inhibiting the expression of a target gene, such as those of formula (XXXVI):

-   -   wherein L^(5A), L^(5B) and L^(5C) represent a monosaccharide,         such as GalNAc derivative.

Examples of suitable bivalent and trivalent branched linker groups conjugating GalNAc derivatives include, but are not limited to, the structures recited above as formulas II, VII, XI, X, and XIII.

Representative U.S. patents that teach the preparation of RNA conjugates include, but are not limited to, U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941; 6,294,664; 6,320,017; 6,576,752; 6,783,931; 6,900,297; 7,037,646; 8,106,022, the entire contents of each of which are hereby incorporated herein by reference.

It is not necessary for all positions in a given compound to be uniformly modified, and in fact more than one of the aforementioned modifications can be incorporated in a single compound or even at a single nucleoside within an iRNA. The present invention also includes iRNA compounds that are chimeric compounds.

“Chimeric” iRNA compounds or “chimeras,” in the context of this invention, are iRNA compounds, preferably dsRNAs, which contain two or more chemically distinct regions, each made up of at least one monomer unit, i.e., a nucleotide in the case of a dsRNA compound. These iRNAs typically contain at least one region wherein the RNA is modified so as to confer upon the iRNA increased resistance to nuclease degradation, increased cellular uptake, and/or increased binding affinity for the target nucleic acid. An additional region of the iRNA can serve as a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNase H is a cellular endonuclease which cleaves the RNA strand of an RNA:DNA duplex. Activation of RNase H, therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of iRNA inhibition of gene expression. Consequently, comparable results can often be obtained with shorter iRNAs when chimeric dsRNAs are used, compared to phosphorothioate deoxy dsRNAs hybridizing to the same target region. Cleavage of the RNA target can be routinely detected by gel electrophoresis and, if necessary, associated nucleic acid hybridization techniques known in the art.

In certain instances, the RNA of an iRNA can be modified by a non-ligand group. A number of non-ligand molecules have been conjugated to iRNAs in order to enhance the activity, cellular distribution or cellular uptake of the iRNA, and procedures for performing such conjugations are available in the scientific literature. Such non-ligand moieties have included lipid moieties, such as cholesterol (Kubo, T. et al., Biochem. Biophys. Res. Comm., 2007, 365(1):54-61; Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86:6553), cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4:1053), a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660:306; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3:2765), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10:111; Kabanov et al., FEBS Lett., 1990, 259:327; Svinarchuk et al., Biochimie, 1993, 75:49), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651; Shea et al., Nucl. Acids Res., 1990, 18:3777), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229), or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923). Representative United States patents that teach the preparation of such RNA conjugates have been listed above. Typical conjugation protocols involve the synthesis of an RNAs bearing an amino linker at one or more positions of the sequence. The amino group is then reacted with the molecule being conjugated using appropriate coupling or activating reagents. The conjugation reaction can be performed either with the RNA still bound to the solid support or following cleavage of the RNA, in solution phase. Purification of the RNA conjugate by HPLC typically affords the pure conjugate.

IV. Delivery of an iRNA of the Invention

The delivery of an iRNA of the invention to a cell e.g., a cell within a subject, such as a human subject (e.g., a subject in need thereof, such as a subject having a glycogen storage disease (GSD), e.g., type Ia GSD) can be achieved in a number of different ways. For example, delivery may be performed by contacting a cell with an iRNA of the invention either in vitro or in vivo. In vivo delivery may also be performed directly by administering a composition comprising an iRNA, e.g., a dsRNA, to a subject. Alternatively, in vivo delivery may be performed indirectly by administering one or more vectors that encode and direct the expression of the iRNA. These alternatives are discussed further below.

In general, any method of delivering a nucleic acid molecule (in vitro or in vivo) can be adapted for use with an iRNA of the invention (see e.g., Akhtar S. and Julian RL., (1992) Trends Cell. Biol. 2(5):139-144 and WO94/02595, which are incorporated herein by reference in their entireties). For in vivo delivery, factors to consider in order to deliver an iRNA molecule include, for example, biological stability of the delivered molecule, prevention of non-specific effects, and accumulation of the delivered molecule in the target tissue. The non-specific effects of an iRNA can be minimized by local administration, for example, by direct injection or implantation into a tissue or topically administering the preparation. Local administration to a treatment site maximizes local concentration of the agent, limits the exposure of the agent to systemic tissues that can otherwise be harmed by the agent or that can degrade the agent, and permits a lower total dose of the iRNA molecule to be administered. Several studies have shown successful knockdown of gene products when an iRNA is administered locally. For example, intraocular delivery of a VEGF dsRNA by intravitreal injection in cynomolgus monkeys (Tolentino, M J. et al., (2004) Retina 24:132-138) and subretinal injections in mice (Reich, S J. et al. (2003) Mol. Vis. 9:210-216) were both shown to prevent neovascularization in an experimental model of age-related macular degeneration. In addition, direct intratumoral injection of a dsRNA in mice reduces tumor volume (Pille, J. et al. (2005) Mol. Ther. 11:267-274) and can prolong survival of tumor-bearing mice (Kim, W J. et al., (2006) Mol. Ther. 14:343-350; Li, S. et al., (2007) Mol. Ther. 15:515-523). RNA interference has also shown success with local delivery to the CNS by direct injection (Dorn, G. et al., (2004) Nucleic Acids 32:e49; Tan, P H. et al. (2005) Gene Ther. 12:59-66; Makimura, H. et al. (2002) BMC Neurosci. 3:18; Shishkina, G T., et al. (2004) Neuroscience 129:521-528; Thakker, E R., et al. (2004) Proc. Natl. Acad. Sci. U.S.A. 101:17270-17275; Akaneya, Y., et al. (2005) J. Neurophysiol. 93:594-602) and to the lungs by intranasal administration (Howard, K A. et al., (2006) Mol. Ther. 14:476-484; Zhang, X. et al., (2004) J. Biol. Chem. 279:10677-10684; Bitko, V. et al., (2005) Nat. Med. 11:50-55). For administering an iRNA systemically for the treatment of a disease, the RNA can be modified or alternatively delivered using a drug delivery system; both methods act to prevent the rapid degradation of the dsRNA by endo- and exo-nucleases in vivo. Modification of the RNA or the pharmaceutical carrier can also permit targeting of the iRNA composition to the target tissue and avoid undesirable off-target effects. iRNA molecules can be modified by chemical conjugation to lipophilic groups such as cholesterol to enhance cellular uptake and prevent degradation. For example, an iRNA directed against ApoB conjugated to a lipophilic cholesterol moiety was injected systemically into mice and resulted in knockdown of apoB mRNA in both the liver and jejunum (Soutschek, J. et al., (2004) Nature 432:173-178). Conjugation of an iRNA to an aptamer has been shown to inhibit tumor growth and mediate tumor regression in a mouse model of prostate cancer (McNamara, JO. et al., (2006) Nat. Biotechnol. 24:1005-1015). In an alternative embodiment, the iRNA can be delivered using drug delivery systems such as a nanoparticle, a dendrimer, a polymer, liposomes, or a cationic delivery system. Positively charged cationic delivery systems facilitate binding of an iRNA molecule (negatively charged) and also enhance interactions at the negatively charged cell membrane to permit efficient uptake of an iRNA by the cell. Cationic lipids, dendrimers, or polymers can either be bound to an iRNA, or induced to form a vesicle or micelle (see e.g., Kim SH. et al., (2008) Journal of Controlled Release 129(2):107-116) that encases an iRNA. The formation of vesicles or micelles further prevents degradation of the iRNA when administered systemically. Methods for making and administering cationic-iRNA complexes are well within the abilities of one skilled in the art (see e.g., Sorensen, D R., et al. (2003) J. Mol. Biol 327:761-766; Verma, U N. et al., (2003) Clin. Cancer Res. 9:1291-1300; Arnold, A S et al., (2007) J. Hypertens. 25:197-205, which are incorporated herein by reference in their entirety). Some non-limiting examples of drug delivery systems useful for systemic delivery of iRNAs include DOTAP (Sorensen, D R., et al (2003), supra; Verma, U N. et al., (2003), supra), Oligofectamine, “solid nucleic acid lipid particles” (Zimmermann, T S. et al., (2006) Nature 441:111-114), cardiolipin (Chien, P Y. et al., (2005) Cancer Gene Ther. 12:321-328; Pal, A. et al., (2005) Int J. Oncol. 26:1087-1091), polyethyleneimine (Bonnet M E. et al., (2008) Pharm. Res. August 16 Epub ahead of print; Aigner, A. (2006) J. Biomed. Biotechnol. 71659), Arg-Gly-Asp (RGD) peptides (Liu, S. (2006) Mol. Pharm. 3:472-487), and polyamidoamines (Tomalia, D A. et al., (2007) Biochem. Soc. Trans. 35:61-67; Yoo, H. et al., (1999) Pharm. Res. 16:1799-1804). In some embodiments, an iRNA forms a complex with cyclodextrin for systemic administration. Methods for administration and pharmaceutical compositions of iRNAs and cyclodextrins can be found in U.S. Pat. No. 7,427,605, which is herein incorporated by reference in its entirety.

A. Vector Encoded iRNAs of the Invention

iRNA targeting the GCK gene can be expressed from transcription units inserted into DNA or RNA vectors (see, e.g., Couture, A, et al., TIG. (1996), 12:5-10; Skillern, A., et al., International PCT Publication No. WO 00/22113, Conrad, International PCT Publication No. WO 00/22114, and Conrad, U.S. Pat. No. 6,054,299). Expression can be transient (on the order of hours to weeks) or sustained (weeks to months or longer), depending upon the specific construct used and the target tissue or cell type. These transgenes can be introduced as a linear construct, a circular plasmid, or a viral vector, which can be an integrating or non-integrating vector. The transgene can also be constructed to permit it to be inherited as an extrachromosomal plasmid (Gassmann, et al., (1995) Proc. Natl. Acad. Sci. USA 92:1292).

The individual strand or strands of an iRNA can be transcribed from a promoter on an expression vector. Where two separate strands are to be expressed to generate, for example, a dsRNA, two separate expression vectors can be co-introduced (e.g., by transfection or infection) into a target cell. Alternatively each individual strand of a dsRNA can be transcribed by promoters both of which are located on the same expression plasmid. In one embodiment, a dsRNA is expressed as inverted repeat polynucleotides joined by a linker polynucleotide sequence such that the dsRNA has a stem and loop structure.

iRNA expression vectors are generally DNA plasmids or viral vectors. Expression vectors compatible with eukaryotic cells, preferably those compatible with vertebrate cells, can be used to produce recombinant constructs for the expression of an iRNA as described herein. Eukaryotic cell expression vectors are well known in the art and are available from a number of commercial sources. Typically, such vectors are provided containing convenient restriction sites for insertion of the desired nucleic acid segment. Delivery of iRNA expressing vectors can be systemic, such as by intravenous or intramuscular administration, by administration to target cells ex-planted from the patient followed by reintroduction into the patient, or by any other means that allows for introduction into a desired target cell.

Viral vector systems which can be utilized with the methods and compositions described herein include, but are not limited to, (a) adenovirus vectors; (b) retrovirus vectors, including but not limited to lentiviral vectors, moloney murine leukemia virus, etc.; (c) adeno-associated virus vectors; (d) herpes simplex virus vectors; (e) SV 40 vectors; (f) polyoma virus vectors; (g) papilloma virus vectors; (h) picornavirus vectors; (i) pox virus vectors such as an orthopox, e.g., vaccinia virus vectors or avipox, e.g. canary pox or fowl pox; and (j) a helper-dependent or gutless adenovirus. Replication-defective viruses can also be advantageous. Different vectors will or will not become incorporated into the cells' genome. The constructs can include viral sequences for transfection, if desired. Alternatively, the construct can be incorporated into vectors capable of episomal replication, e.g. EPV and EBV vectors. Constructs for the recombinant expression of an iRNA will generally require regulatory elements, e.g., promoters, enhancers, etc., to ensure the expression of the iRNA in target cells. Other aspects to consider for vectors and constructs are known in the art.

V. Pharmaceutical Compositions of the Invention

The present invention also includes pharmaceutical compositions and formulations which include the iRNAs of the invention. In one embodiment, provided herein are pharmaceutical compositions containing an iRNA, as described herein, and a pharmaceutically acceptable carrier. The pharmaceutical compositions containing the iRNA are useful for treating a disease or disorder associated with the expression or activity of a GCK gene, such as, a glycogen storage disease (GSD), e.g., type Ia GSD, or one or more signs or symptoms of GSD, such as hypoglycemia, lactic acidosis, hyperuricemia, hyperlipidemia, hepatomegaly, kidney disease as a result of glycogen accumulation, hunger, jitteriness, lethargy, apnea, seizures, diaphoresis, confusion, headaches, dizziness, unusual mood or behavior changes, loss of consciousness, coma, muscle cramps, bleeding diathesis, short stauture, osteoporosis, delayed puberty, gout, renal disease, systemic hypertension, pulmonary hypertension, hepatic adenomas, pancreatitis, anemia, vitamin D deficiency, polycystic ovaries, irregular menstrual cycles, menorrhagia, and eruptive xanthomata.

Such pharmaceutical compositions are formulated based on the mode of delivery. One example is compositions that are formulated for systemic administration via parenteral delivery, e.g., by intravenous (IV) or for subcutaneous delivery. Another example is compositions that are formulated for direct delivery into the liver, e.g., by infusion into the liver, such as by continuous pump infusion.

The pharmaceutical compositions of the invention may be administered in dosages sufficient to inhibit expression of a GCK gene. In general, a suitable dose of an iRNA of the invention will be in the range of about 0.001 to about 200.0 milligrams per kilogram body weight of the recipient per day, generally in the range of about 1 to 50 mg per kilogram body weight per day. Typically, a suitable dose of an iRNA of the invention will be in the range of about 0.1 mg/kg to about 5.0 mg/kg, preferably about 0.3 mg/kg and about 3.0 mg/kg. A repeat-dose regimine may include administration of a therapeutic amount of iRNA on a regular basis, such as every other day or once a year. In certain embodiments, the iRNA is administered about once per month to about once per quarter (i.e., about once every three months).

After an initial treatment regimen, the treatments can be administered on a less frequent basis.

The skilled artisan will appreciate that certain factors can influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of a composition can include a single treatment or a series of treatments. Estimates of effective dosages and in vivo half-lives for the individual iRNAs encompassed by the invention can be made using conventional methodologies or on the basis of in vivo testing using an appropriate animal model, as described elsewhere herein.

Advances in mouse genetics have generated a number of mouse models for the study of various human diseases, such as disorders of excess glucose that would benefit from reduction in the expression of GCK.

The pharmaceutical compositions of the present invention can be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration can be topical (e.g., by a transdermal patch), pulmonary, e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal, oral or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; subdermal, e.g., via an implanted device; or intracranial, e.g., by intraparenchymal, intrathecal or intraventricular, administration.

The iRNA can be delivered in a manner to target a particular tissue, such as the liver (e.g., the hepatocytes of the liver).

Pharmaceutical compositions and formulations for topical administration can include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids, and powders. Conventional pharmaceutical carriers, aqueous, powder, or oily bases, thickeners and the like can be necessary or desirable. Coated condoms, gloves and the like can also be useful. Suitable topical formulations include those in which the iRNAs featured in the invention are in admixture with a topical delivery agent such as lipids, liposomes, fatty acids, fatty acid esters, steroids, chelating agents, and surfactants. Suitable lipids and liposomes include neutral (e.g., dioleoylphosphatidyl DOPE ethanolamine, dimyristoylphosphatidyl choline DMPC, distearolyphosphatidyl choline), negative (e.g., dimyristoylphosphatidyl glycerol DMPG), and cationic (e.g., dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidyl ethanolamine DOTMA). iRNAs featured in the invention can be encapsulated within liposomes or can form complexes thereto, in particular to cationic liposomes. Alternatively, iRNAs can be complexed to lipids, in particular to cationic lipids. Suitable fatty acids and esters include but are not limited to arachidonic acid, oleic acid, eicosanoic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or a C₁₋₂₀ alkyl ester (e.g., isopropylmyristate IPM), monoglyceride or diglyceride; or pharmaceutically acceptable salt thereof. Topical formulations are described in detail in U.S. Pat. No. 6,747,014, which is incorporated herein by reference.

A. iRNA Formulations Comprising Membranous Molecular Assemblies

An iRNA for use in the compositions and methods of the invention can be formulated for delivery in a membranous molecular assembly, e.g., a liposome or a micelle. As used herein, the term “liposome” refers to a vesicle composed of amphiphilic lipids arranged in at least one bilayer, e.g., one bilayer or a plurality of bilayers. Liposomes include unilamellar and multilamellar vesicles that have a membrane formed from a lipophilic material and an aqueous interior. The aqueous portion contains the iRNA composition. The lipophilic material isolates the aqueous interior from an aqueous exterior, which typically does not include the iRNA composition, although in some examples, it may. Liposomes are useful for the transfer and delivery of active ingredients to the site of action. Because the liposomal membrane is structurally similar to biological membranes, when liposomes are applied to a tissue, the liposomal bilayer fuses with bilayer of the cellular membranes. As the merging of the liposome and cell progresses, the internal aqueous contents that include the iRNA are delivered into the cell where the iRNA can specifically bind to a target RNA and can mediate RNAi. In some cases the liposomes are also specifically targeted, e.g., to direct the iRNA to particular cell types.

A liposome containing a RNAi agent can be prepared by a variety of methods. In one example, the lipid component of a liposome is dissolved in a detergent so that micelles are formed with the lipid component. For example, the lipid component can be an amphipathic cationic lipid or lipid conjugate. The detergent can have a high critical micelle concentration and may be nonionic. Exemplary detergents include cholate, CHAPS, octylglucoside, deoxycholate, and lauroyl sarcosine. The RNAi agent preparation is then added to the micelles that include the lipid component. The cationic groups on the lipid interact with the RNAi agent and condense around the RNAi agent to form a liposome. After condensation, the detergent is removed, e.g., by dialysis, to yield a liposomal preparation of RNAi agent.

If necessary a carrier compound that assists in condensation can be added during the condensation reaction, e.g., by controlled addition. For example, the carrier compound can be a polymer other than a nucleic acid (e.g., spermine or spermidine). pH can also adjusted to favor condensation.

Methods for producing stable polynucleotide delivery vehicles, which incorporate a polynucleotide/cationic lipid complex as structural components of the delivery vehicle, are further described in, e.g., WO 96/37194, the entire contents of which are incorporated herein by reference. Liposome formation can also include one or more aspects of exemplary methods described in Felgner, P. L. et al., (1987) Proc. Natl. Acad. Sci. USA 8:7413-7417; U.S. Pat. Nos. 4,897,355; 5,171,678; Bangham et al., (1965) M. Mol. Biol. 23:238; Olson et al., (1979) Biochim. Biophys. Acta 557:9; Szoka et al., (1978) Proc. Natl. Acad. Sci. 75: 4194; Mayhew et al., (1984) Biochim. Biophys. Acta 775:169; Kim et al., (1983) Biochim. Biophys. Acta 728:339; and Fukunaga et al., (1984) Endocrinol. 115:757. Commonly used techniques for preparing lipid aggregates of appropriate size for use as delivery vehicles include sonication and freeze-thaw plus extrusion (see, e.g., Mayer et al., (1986) Biochim. Biophys. Acta 858:161. Microfluidization can be used when consistently small (50 to 200 nm) and relatively uniform aggregates are desired (Mayhew et al., (1984) Biochim. Biophys. Acta 775:169. These methods are readily adapted to packaging RNAi agent preparations into liposomes.

Liposomes fall into two broad classes. Cationic liposomes are positively charged liposomes which interact with the negatively charged nucleic acid molecules to form a stable complex. The positively charged nucleic acid/liposome complex binds to the negatively charged cell surface and is internalized in an endosome. Due to the acidic pH within the endosome, the liposomes are ruptured, releasing their contents into the cell cytoplasm (Wang et al. (1987) Biochem. Biophys. Res. Commun., 147:980-985).

Liposomes, which are pH-sensitive or negatively charged, entrap nucleic acids rather than complex with them. Since both the nucleic acid and the lipid are similarly charged, repulsion rather than complex formation occurs. Nevertheless, some nucleic acid is entrapped within the aqueous interior of these liposomes. pH sensitive liposomes have been used to deliver nucleic acids encoding the thymidine kinase gene to cell monolayers in culture. Expression of the exogenous gene was detected in the target cells (Zhou et al. (1992) Journal of Controlled Release, 19:269-274).

One major type of liposomal composition includes phospholipids other than naturally-derived phosphatidylcholine. Neutral liposome compositions, for example, can be formed from dimyristoyl phosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC). Anionic liposome compositions generally are formed from dimyristoyl phosphatidylglycerol, while anionic fusogenic liposomes are formed primarily from dioleoyl phosphatidylethanolamine (DOPE). Another type of liposomal composition is formed from phosphatidylcholine (PC) such as, for example, soybean PC, and egg PC. Another type is formed from mixtures of phospholipid and/or phosphatidylcholine and/or cholesterol.

Examples of other methods to introduce liposomes into cells in vitro and in vivo include U.S. Pat. Nos. 5,283,185; 5,171,678; WO 94/00569; WO 93/24640; WO 91/16024; Felgner, (1994) J. Biol. Chem. 269:2550; Nabel, (1993) Proc. Natl. Acad. Sci. 90:11307; Nabel, (1992) Human Gene Ther. 3:649; Gershon, (1993) Biochem. 32:7143; and Strauss, (1992) EMBO J. 11:417.

Non-ionic liposomal systems have also been examined to determine their utility in the delivery of drugs to the skin, in particular systems comprising non-ionic surfactant and cholesterol. Non-ionic liposomal formulations comprising Novasome™ I (glyceryl dilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome™ II (glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) were used to deliver cyclosporin-A into the dermis of mouse skin. Results indicated that such non-ionic liposomal systems were effective in facilitating the deposition of cyclosporine A into different layers of the skin (Hu et al., (1994) S.T.P.Pharma. Sci., 4(6):466).

Liposomes also include “sterically stabilized” liposomes, a term which, as used herein, refers to liposomes comprising one or more specialized lipids that, when incorporated into liposomes, result in enhanced circulation lifetimes relative to liposomes lacking such specialized lipids. Examples of sterically stabilized liposomes are those in which part of the vesicle-forming lipid portion of the liposome (A) comprises one or more glycolipids, such as monosialoganglioside G_(M1), or (B) is derivatized with one or more hydrophilic polymers, such as a polyethylene glycol (PEG) moiety. While not wishing to be bound by any particular theory, it is thought in the art that, at least for sterically stabilized liposomes containing gangliosides, sphingomyelin, or PEG-derivatized lipids, the enhanced circulation half-life of these sterically stabilized liposomes derives from a reduced uptake into cells of the reticuloendothelial system (RES) (Allen et al., (1987) FEBS Letters, 223:42; Wu et al., (1993) Cancer Research, 53:3765).

Various liposomes comprising one or more glycolipids are known in the art. Papahadjopoulos et al. (Ann. N.Y. Acad. Sci., (1987), 507:64) reported the ability of monosialoganglioside G_(M1), galactocerebroside sulfate and phosphatidylinositol to improve blood half-lives of liposomes. These findings were expounded upon by Gabizon et al. (Proc. Natl. Acad. Sci. U.S.A., (1988), 85:6949). U.S. Pat. No. 4,837,028 and WO 88/04924, both to Allen et al., disclose liposomes comprising (1) sphingomyelin and (2) the ganglioside G_(M1) or a galactocerebroside sulfate ester. U.S. Pat. No. 5,543,152 (Webb et al.) discloses liposomes comprising sphingomyelin. Liposomes comprising 1,2-sn-dimyristoylphosphatidylcholine are disclosed in WO 97/13499 (Lim et al).

In one embodiment, cationic liposomes are used. Cationic liposomes possess the advantage of being able to fuse to the cell membrane. Non-cationic liposomes, although not able to fuse as efficiently with the plasma membrane, are taken up by macrophages in vivo and can be used to deliver RNAi agents to macrophages.

Further advantages of liposomes include: liposomes obtained from natural phospholipids are biocompatible and biodegradable; liposomes can incorporate a wide range of water and lipid soluble drugs; liposomes can protect encapsulated RNAi agents in their internal compartments from metabolism and degradation (Rosoff, in “Pharmaceutical Dosage Forms,” Lieberman, Rieger and Banker (Eds.), 1988, volume 1, p. 245). Important considerations in the preparation of liposome formulations are the lipid surface charge, vesicle size and the aqueous volume of the liposomes.

A positively charged synthetic cationic lipid, N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA) can be used to form small liposomes that interact spontaneously with nucleic acid to form lipid-nucleic acid complexes which are capable of fusing with the negatively charged lipids of the cell membranes of tissue culture cells, resulting in delivery of RNAi agent (see, e.g., Felgner, P. L. et al., (1987) Proc. Natl. Acad. Sci. USA 8:7413-7417, and U.S. Pat. No. 4,897,355 for a description of DOTMA and its use with DNA).

A DOTMA analogue, 1,2-bis(oleoyloxy)-3-(trimethylammonia)propane (DOTAP) can be used in combination with a phospholipid to form DNA-complexing vesicles. Lipofectin™ (Bethesda Research Laboratories, Gaithersburg, Md.) is an effective agent for the delivery of highly anionic nucleic acids into living tissue culture cells that comprise positively charged DOTMA liposomes which interact spontaneously with negatively charged polynucleotides to form complexes. When enough positively charged liposomes are used, the net charge on the resulting complexes is also positive. Positively charged complexes prepared in this way spontaneously attach to negatively charged cell surfaces, fuse with the plasma membrane, and efficiently deliver functional nucleic acids into, for example, tissue culture cells. Another commercially available cationic lipid, 1,2-bis(oleoyloxy)-3,3-(trimethylammonia)propane (“DOTAP”) (Boehringer Mannheim™, Indianapolis, Ind.) differs from DOTMA in that the oleoyl moieties are linked by ester, rather than ether linkages.

Other reported cationic lipid compounds include those that have been conjugated to a variety of moieties including, for example, carboxyspermine which has been conjugated to one of two types of lipids and includes compounds such as 5-carboxyspermylglycine dioctaoleoylamide (“DOGS”) (Transfectam™, Promega™, Madison, Wis.) and dipalmitoylphosphatidylethanolamine 5-carboxyspermyl-amide (“DPPES”) (see, e.g., U.S. Pat. No. 5,171,678).

Another cationic lipid conjugate includes derivatization of the lipid with cholesterol (“DC-Chol”) which has been formulated into liposomes in combination with DOPE (See, Gao, X. and Huang, L., (1991) Biochim. Biophys. Res. Commun. 179:280). Lipopolylysine, made by conjugating polylysine to DOPE, has been reported to be effective for transfection in the presence of serum (Zhou, X. et al., (1991) Biochim. Biophys. Acta 1065:8). For certain cell lines, these liposomes containing conjugated cationic lipids, are said to exhibit lower toxicity and provide more efficient transfection than the DOTMA-containing compositions. Other commercially available cationic lipid products include DMRIE and DMRIE-HP (Vical™, La Jolla, Calif.) and Lipofectamine™ (DOSPA) (Life Technology™, Inc., Gaithersburg, Md.). Other cationic lipids suitable for the delivery of oligonucleotides are described in WO 98/39359 and WO 96/37194.

Liposomal formulations are particularly suited for topical administration, liposomes present several advantages over other formulations. Such advantages include reduced side effects related to high systemic absorption of the administered drug, increased accumulation of the administered drug at the desired target, and the ability to administer RNAi agent into the skin. In some implementations, liposomes are used for delivering RNAi agent to epidermal cells and also to enhance the penetration of RNAi agent into dermal tissues, e.g., into skin. For example, the liposomes can be applied topically. Topical delivery of drugs formulated as liposomes to the skin has been documented (see, e.g., Weiner et al., (1992) Journal of Drug Targeting, vol. 2,405-410 and du Plessis et al., (1992) Antiviral Research, 18:259-265; Mannino, R. J. and Fould-Fogerite, S., (1998) Biotechniques 6:682-690; Itani, T. et al., (1987) Gene 56:267-276; Nicolau, C. et al. (1987) Meth. Enzymol. 149:157-176; Straubinger, R. M. and Papahadjopoulos, D. (1983) Meth. Enzymol. 101:512-527; Wang, C. Y. and Huang, L., (1987) Proc. Natl. Acad. Sci. USA 84:7851-7855).

Non-ionic liposomal systems have also been examined to determine their utility in the delivery of drugs to the skin, in particular systems comprising non-ionic surfactant and cholesterol. Non-ionic liposomal formulations comprising Novasome I (glyceryl dilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome II (glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) were used to deliver a drug into the dermis of mouse skin. Such formulations with RNAi agent are useful for treating a dermatological disorder.

Liposomes that include iRNA can be made highly deformable. Such deformability can enable the liposomes to penetrate through pore that are smaller than the average radius of the liposome. For example, transfersomes are a type of deformable liposomes. Transferosomes can be made by adding surface edge activators, usually surfactants, to a standard liposomal composition. Transfersomes that include RNAi agent can be delivered, for example, subcutaneously by infection in order to deliver RNAi agent to keratinocytes in the skin. In order to cross intact mammalian skin, lipid vesicles must pass through a series of fine pores, each with a diameter less than 50 nm, under the influence of a suitable transdermal gradient. In addition, due to the lipid properties, these transferosomes can be self-optimizing (adaptive to the shape of pores, e.g., in the skin), self-repairing, and can frequently reach their targets without fragmenting, and often self-loading. Transfersomes have been used to deliver serum albumin to the skin. The transfersome-mediated delivery of serum albumin has been shown to be as effective as subcutaneous injection of a solution containing serum albumin.

Other formulations amenable to the present invention are described in, for example, PCT Publication No. WO 2008/042973.

Transfersomes are yet another type of liposomes, and are highly deformable lipid aggregates which are attractive candidates for drug delivery vehicles. Transfersomes can be described as lipid droplets which are so highly deformable that they are easily able to penetrate through pores which are smaller than the droplet. Transfersomes are adaptable to the environment in which they are used, e.g., they are self-optimizing (adaptive to the shape of pores in the skin), self-repairing, frequently reach their targets without fragmenting, and often self-loading. To make transfersomes it is possible to add surface edge-activators, usually surfactants, to a standard liposomal composition. Transfersomes have been used to deliver serum albumin to the skin. The transfersome-mediated delivery of serum albumin has been shown to be as effective as subcutaneous injection of a solution containing serum albumin.

Surfactants find wide application in formulations such as emulsions (including microemulsions) and liposomes. The most common way of classifying and ranking the properties of the many different types of surfactants, both natural and synthetic, is by the use of the hydrophile/lipophile balance (HLB). The nature of the hydrophilic group (also known as the “head”) provides the most useful means for categorizing the different surfactants used in formulations (Rieger, in Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).

If the surfactant molecule is not ionized, it is classified as a nonionic surfactant. Nonionic surfactants find wide application in pharmaceutical and cosmetic products and are usable over a wide range of pH values. In general their HLB values range from 2 to about 18 depending on their structure. Nonionic surfactants include nonionic esters such as ethylene glycol esters, propylene glycol esters, glyceryl esters, polyglyceryl esters, sorbitan esters, sucrose esters, and ethoxylated esters. Nonionic alkanolamides and ethers such as fatty alcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylated block polymers are also included in this class. The polyoxyethylene surfactants are the most popular members of the nonionic surfactant class.

If the surfactant molecule carries a negative charge when it is dissolved or dispersed in water, the surfactant is classified as anionic. Anionic surfactants include carboxylates such as soaps, acyl lactylates, acyl amides of amino acids, esters of sulfuric acid such as alkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkyl benzene sulfonates, acyl isethionates, acyl taurates and sulfosuccinates, and phosphates. The most important members of the anionic surfactant class are the alkyl sulfates and the soaps.

If the surfactant molecule carries a positive charge when it is dissolved or dispersed in water, the surfactant is classified as cationic. Cationic surfactants include quaternary ammonium salts and ethoxylated amines. The quaternary ammonium salts are the most used members of this class.

If the surfactant molecule has the ability to carry either a positive or negative charge, the surfactant is classified as amphoteric. Amphoteric surfactants include acrylic acid derivatives, substituted alkylamides, N-alkylbetaines, and phosphatides.

The use of surfactants in drug products, formulations and in emulsions has been reviewed (Rieger, in Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).

The iRNA for use in the methods of the invention can also be provided as micellar formulations. “Micelles” are defined herein as a particular type of molecular assembly in which amphipathic molecules are arranged in a spherical structure such that all the hydrophobic portions of the molecules are directed inward, leaving the hydrophilic portions in contact with the surrounding aqueous phase. The converse arrangement exists if the environment is hydrophobic.

A mixed micellar formulation suitable for delivery through transdermal membranes may be prepared by mixing an aqueous solution of the siRNA composition, an alkali metal C₈ to C₂₂ alkyl sulphate, and a micelle forming compounds. Exemplary micelle forming compounds include lecithin, hyaluronic acid, pharmaceutically acceptable salts of hyaluronic acid, glycolic acid, lactic acid, chamomile extract, cucumber extract, oleic acid, linoleic acid, linolenic acid, monoolein, monooleates, monolaurates, borage oil, evening of primrose oil, menthol, trihydroxy oxo cholanyl glycine and pharmaceutically acceptable salts thereof, glycerin, polyglycerin, lysine, polylysine, triolein, polyoxyethylene ethers and analogues thereof, polidocanol alkyl ethers and analogues thereof, chenodeoxycholate, deoxycholate, and mixtures thereof. The micelle forming compounds may be added at the same time or after addition of the alkali metal alkyl sulphate. Mixed micelles will form with substantially any kind of mixing of the ingredients but vigorous mixing in order to provide smaller size micelles.

In one method a first micellar composition is prepared which contains the siRNA composition and at least the alkali metal alkyl sulphate. The first micellar composition is then mixed with at least three micelle forming compounds to form a mixed micellar composition. In another method, the micellar composition is prepared by mixing the siRNA composition, the alkali metal alkyl sulphate and at least one of the micelle forming compounds, followed by addition of the remaining micelle forming compounds, with vigorous mixing.

Phenol and/or m-cresol may be added to the mixed micellar composition to stabilize the formulation and protect against bacterial growth. Alternatively, phenol and/or m-cresol may be added with the micelle forming ingredients. An isotonic agent such as glycerin may also be added after formation of the mixed micellar composition.

For delivery of the micellar formulation as a spray, the formulation can be put into an aerosol dispenser and the dispenser is charged with a propellant. The propellant, which is under pressure, is in liquid form in the dispenser. The ratios of the ingredients are adjusted so that the aqueous and propellant phases become one, i.e., there is one phase. If there are two phases, it is necessary to shake the dispenser prior to dispensing a portion of the contents, e.g., through a metered valve. The dispensed dose of pharmaceutical agent is propelled from the metered valve in a fine spray.

Propellants may include hydrogen-containing chlorofluorocarbons, hydrogen-containing fluorocarbons, dimethyl ether, and diethyl ether. In certain embodiments, HFA 134a (1,1,1,2 tetrafluoroethane) may be used.

The specific concentrations of the essential ingredients can be determined by relatively straightforward experimentation. For absorption through the oral cavities, it is often desirable to increase, e.g., at least double or triple, the dosage for through injection or administration through the gastrointestinal tract.

B. Lipid Particles

iRNAs, e.g., dsRNAs of in the invention may be fully encapsulated in a lipid formulation, e.g., a LNP, or other nucleic acid-lipid particle.

As used herein, the term “LNP” refers to a stable nucleic acid-lipid particle. LNPs typically contain a cationic lipid, a non-cationic lipid, and a lipid that prevents aggregation of the particle (e.g., a PEG-lipid conjugate). LNPs are extremely useful for systemic applications, as they exhibit extended circulation lifetimes following intravenous (i.v.) injection and accumulate at distal sites (e.g., sites physically separated from the administration site). LNPs include “pSPLP,” which include an encapsulated condensing agent-nucleic acid complex as set forth in PCT Publication No. WO 00/03683. The particles of the present invention typically have a mean diameter of about 50 nm to about 150 nm, more typically about 60 nm to about 130 nm, more typically about 70 nm to about 110 nm, most typically about 70 nm to about 90 nm, and are substantially nontoxic. In addition, the nucleic acids when present in the nucleic acid-lipid particles of the present invention are resistant in aqueous solution to degradation with a nuclease. Nucleic acid-lipid particles and their method of preparation are disclosed in, e.g., U.S. Pat. Nos. 5,976,567; 5,981,501; 6,534,484; 6,586,410; 6,815,432; U.S. Publication No. 2010/0324120 and PCT Publication No. WO 96/40964.

In one embodiment, the lipid to drug ratio (mass/mass ratio) (e.g., lipid to dsRNA ratio) will be in the range of from about 1:1 to about 50:1, from about 1:1 to about 25:1, from about 3:1 to about 15:1, from about 4:1 to about 10:1, from about 5:1 to about 9:1, or about 6:1 to about 9:1. Ranges intermediate to the above recited ranges are also contemplated to be part of the invention.

The cationic lipid can be, for example, N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N—(I-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), N—(I-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA), 1,2-DiLinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), 1,2-Dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP), 1,2-Dilinoleyoxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-Dilinoleyoxy-3-morpholinopropane (DLin-MA), 1,2-Dilinoleoyl-3-dimethylaminopropane (DLinDAP), 1,2-Dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), 1-Linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-Dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl), 1,2-Dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.Cl), 1,2-Dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), or 3-(N,N-Dilinoleylamino)-1,2-propanediol (DLinAP), 3-(N,N-Dioleylamino)-1,2-propanedio (DOAP), 1,2-Dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), 1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA), 2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA) or analogs thereof, (3aR,5s,6aS)—N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine (ALN100), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (MC3), 1,1′-(2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethylazanediyl)didodecan-2-ol (Tech G1), or a mixture thereof. The cationic lipid can comprise from about 20 mol % to about 50 mol % or about 40 mol % of the total lipid present in the particle.

In another embodiment, the compound 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane can be used to prepare lipid-siRNA nanoparticles. Synthesis of 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane is described in U.S. provisional patent application No. 61/107,998 filed on Oct. 23, 2008, which is herein incorporated by reference.

In one embodiment, the lipid-siRNA particle includes 40% 2, 2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane: 10% DSPC: 40% Cholesterol: 10% PEG-C-DOMG (mole percent) with a particle size of 63.0±20 nm and a 0.027 siRNA/Lipid Ratio.

The ionizable/non-cationic lipid can be an anionic lipid or a neutral lipid including, but not limited to, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), cholesterol; or a mixture thereof. The non-cationic lipid can be from about 5 mol % to about 90 mol %, about 10 mol %, or about 58 mol % if cholesterol is included, of the total lipid present in the particle.

The conjugated lipid that inhibits aggregation of particles can be, for example, a polyethyleneglycol (PEG)-lipid including, without limitation, a PEG-diacylglycerol (DAG), a PEG-dialkyloxypropyl (DAA), a PEG-phospholipid, a PEG-ceramide (Cer), or a mixture thereof. The PEG-DAA conjugate can be, for example, a PEG-dilauryloxypropyl (Ci₂), a PEG-dimyristyloxypropyl (Ci₄), a PEG-dipalmityloxypropyl (Ci6), or a PEG-distearyloxypropyl (C]₈). The conjugated lipid that prevents aggregation of particles can be from 0 mol % to about 20 mol % or about 2 mol % of the total lipid present in the particle.

In some embodiments, the nucleic acid-lipid particle further includes cholesterol at, e.g., about 10 mol % to about 60 mol % or about 48 mol % of the total lipid present in the particle.

In one embodiment, the lipidoid ND98.4HCl (MW 1487) (see US20090023673, filed Mar. 26, 2008, which is incorporated herein by reference), Cholesterol (Sigma-Aldrich™), and PEG-Ceramide C16 (Avanti™ Polar Lipids) can be used to prepare lipid-dsRNA nanoparticles (i.e., LNP01 particles). Stock solutions of each in ethanol can be prepared as follows: ND98, 133 mg/ml; cholesterol, 25 mg/ml, PEG-Ceramide C16, 100 mg/ml. The ND98, cholesterol, and PEG-Ceramide C16 stock solutions can then be combined in a, e.g., 42:48:10 molar ratio. The combined lipid solution can be mixed with aqueous dsRNA (e.g., in sodium acetate pH 5) such that the final ethanol concentration is about 35-45% and the final sodium acetate concentration is about 100-300 mM. Lipid-dsRNA nanoparticles typically form spontaneously upon mixing. Depending on the desired particle size distribution, the resultant nanoparticle mixture can be extruded through a polycarbonate membrane (e.g., 100 nm cut-off) using, for example, a thermobarrel extruder, such as Lipex Extruder (Northern Lipids, Inc). In some cases, the extrusion step can be omitted. Ethanol removal and simultaneous buffer exchange can be accomplished by, for example, dialysis or tangential flow filtration. Buffer can be exchanged with, for example, phosphate buffered saline (PBS) at about pH 7, e.g., about pH 6.9, about pH 7.0, about pH 7.1, about pH 7.2, about pH 7.3, or about pH 7.4.

LNP01 formulations are described, e.g., in International Application Publication No. WO 2008/042973, which is hereby incorporated by reference.

Additional exemplary lipid-dsRNA formulations are described in the table below.

cationic lipid/non-cationic lipid/cholesterol/PEG-lipid conjugate Ionizable/Cationic Lipid Lipid:siRNA ratio SNALP-1 1,2-Dilinolenyloxy-N,N- DLinDMA/DPPC/Cholesterol/PEG- dimethylaminopropane (DLinDMA) cDMA (57.1/7.1/34.4/1.4) lipid:siRNA ~7:1 2-XTC 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]- XTC/DPPC/Cholesterol/PEG- dioxolane (XTC) cDMA 57.1/7.1/34.4/1.4 lipid:siRNA ~7:1 LNP05 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]- XTC/DSPC/Cholesterol/PEG-DMG dioxolane (XTC) 57.5/7.5/31.5/3.5 lipid:siRNA ~6:1 LNP06 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]- XTC/DSPC/Cholesterol/PEG-DMG dioxolane (XTC) 57.5/7.5/31.5/3.5 lipid:siRNA ~11:1 LNP07 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]- XTC/DSPC/Cholesterol/PEG-DMG dioxolane (XTC) 60/7.5/31/1.5, lipid:siRNA ~6:1 LNP08 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]- XTC/DSPC/Cholesterol/PEG-DMG dioxolane (XTC) 60/7.5/31/1.5, lipid:siRNA ~11:1 LNP09 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]- XTC/DSPC/Cholesterol/PEG-DMG dioxolane (XTC) 50/10/38.5/1.5 Lipid:siRNA 10:1 LNP10 (3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)- ALN100/DSPC/Cholesterol/PEG- octadeca-9,12-dienyl)tetrahydro-3aH- DMG cyclopenta[d][1,3]dioxol-5-amine (ALN100) 50/10/38.5/1.5 Lipid:siRNA 10:1 LNP11 (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31- MC-3/DSPC/Cholesterol/PEG- tetraen-19-yl 4-(dimethylamino)butanoate DMG (MC3) 50/10/38.5/1.5 Lipid:siRNA 10:1 LNP12 1,1′-(2-(4-(2-((2-(bis(2- Tech G1/DSPC/Cholesterol/PEG- hydroxydodecyl)amino)ethyl)(2- DMG hydroxydodecyl)amino)ethyl)piperazin-1- 50/10/38.5/1.5 yl)ethylazanediyl)didodecan-2-ol (Tech G1) Lipid:siRNA 10:1 LNP13 XTC XTC/DSPC/Chol/PEG-DMG 50/10/38.5/1.5 Lipid:siRNA: 33:1 LNP14 MC3 MC3/DSPC/Chol/PEG-DMG 40/15/40/5 Lipid:siRNA: 11:1 LNP15 MC3 MC3/DSPC/Chol/PEG- DSG/GalNAc-PEG-DSG 50/10/35/4.5/0.5 Lipid:siRNA: 11:1 LNP16 MC3 MC3/DSPC/Chol/PEG-DMG 50/10/38.5/1.5 Lipid:siRNA: 7:1 LNP17 MC3 MC3/DSPC/Chol/PEG-DSG 50/10/38.5/1.5 Lipid:siRNA: 10:1 LNP18 MC3 MC3/DSPC/Chol/PEG-DMG 50/10/38.5/1.5 Lipid:siRNA: 12:1 LNP19 MC3 MC3/DSPC/Chol/PEG-DMG 50/10/35/5 Lipid:siRNA: 8:1 LNP20 MC3 MC3/DSPC/Chol/PEG-DPG 50/10/38.5/1.5 Lipid:siRNA: 10:1 LNP21 C12-200 C12-200/DSPC/Chol/PEG-DSG 50/10/38.5/1.5 Lipid:siRNA: 7:1 LNP22 XTC XTC/DSPC/Chol/PEG-DSG 50/10/38.5/1.5 Lipid:siRNA: 10:1 DSPC: distearoylphosphatidylcholine DPPC: dipalmitoylphosphatidylcholine PEG-DMG: PEG-didimyristoyl glycerol (C14-PEG, or PEG-C14) (PEG with avg mol wt of 2000) PEG-DSG: PEG-distyryl glycerol (C18-PEG, or PEG-C18) (PEG with avg mol wt of 2000) PEG-cDMA: PEG-carbamoyl-1,2-dimyristyloxypropylamine (PEG with avg mol wt of 2000) SNALP (1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA)) comprising formulations are described in International Publication No. WO2009/127060, filed Apr. 15, 2009, which is hereby incorporated by reference.

XTC comprising formulations are described in, for example, PCT Publication No. WO 2010/088537, the entire contents of which are hereby incorporated herein by reference.

MC3 comprising formulations are described, e.g., in U.S. Publication No. 2010/0324120, filed Jun. 10, 2010, the entire contents of which are hereby incorporated by reference.

ALNY-100 comprising formulations are described in, for example, PCT Publication No. WO 2010/054406, the entire contents of which are hereby incorporated herein by reference.

C12-200 comprising formulations are described in, for example, PCT Publication No. WO 2010/129709, the entire contents of which are hereby incorporated herein by reference.

Compositions and formulations for oral administration include powders or granules, microparticulates, nanoparticulates, suspensions, or solutions in water or non-aqueous media, capsules, gel capsules, sachets, tablets, or minitablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids, or binders can be desirable. In some embodiments, oral formulations are those in which dsRNAs featured in the invention are administered in conjunction with one or more penetration enhancer surfactants and chelators. Suitable surfactants include fatty acids and/or esters or salts thereof, bile acids and/or salts thereof. Suitable bile acids/salts include chenodeoxycholic acid (CDCA) and ursodeoxychenodeoxycholic acid (UDCA), cholic acid, dehydrocholic acid, deoxycholic acid, glucholic acid, glycholic acid, glycodeoxycholic acid, taurocholic acid, taurodeoxycholic acid, sodium tauro-24,25-dihydro-fusidate and sodium glycodihydrofusidate. Suitable fatty acids include arachidonic acid, undecanoic acid, oleic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or a monoglyceride or a diglyceride; or a pharmaceutically acceptable salt thereof (e.g., sodium). In some embodiments, combinations of penetration enhancers are used, for example, fatty acids/salts in combination with bile acids/salts. One exemplary combination is the sodium salt of lauric acid, capric acid and UDCA. Further penetration enhancers include polyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether. DsRNAs featured in the invention can be delivered orally, in granular form including sprayed dried particles, or complexed to form micro or nanoparticles. DsRNA complexing agents include polyamino acids; polyimines; polyacrylates; polyalkylacrylates, polyoxethanes, polyalkylcyanoacrylates; cationized gelatins, albumins, starches, acrylates, polyethyleneglycols (PEG) and starches; polyalkylcyanoacrylates; DEAE-derivatized polyimines, pollulans, celluloses and starches. Suitable complexing agents include chitosan, N-trimethylchitosan, poly-L-lysine, polyhistidine, polyornithine, polyspermines, protamine, polyvinylpyridine, polythiodiethylaminomethylethylene P(TDAE), polyaminostyrene (e.g., p-amino), poly(methylcyanoacrylate), poly(ethylcyanoacrylate), poly(butylcyanoacrylate), poly(isobutylcyanoacrylate), poly(isohexylcynaoacrylate), DEAE-methacrylate, DEAE-hexylacrylate, DEAE-acrylamide, DEAE-albumin and DEAE-dextran, polymethylacrylate, polyhexylacrylate, poly(D,L-lactic acid), poly(DL-lactic-co-glycolic acid (PLGA), alginate, and polyethyleneglycol (PEG). Oral formulations for dsRNAs and their preparation are described in detail in U.S. Pat. No. 6,887,906, US Publn. No. 20030027780, and U.S. Pat. No. 6,747,014, each of which is incorporated herein by reference.

Compositions and formulations for parenteral, intraparenchymal (into the brain), intrathecal, intraventricular, or intrahepatic administration can include sterile aqueous solutions which can also contain buffers, diluents and other suitable additives such as, but not limited to, penetration enhancers, carrier compounds, and other pharmaceutically acceptable carriers or excipients.

Pharmaceutical compositions of the present invention include, but are not limited to, solutions, emulsions, and liposome-containing formulations. These compositions can be generated from a variety of components that include, but are not limited to, preformed liquids, self-emulsifying solids, and self-emulsifying semisolids. Particularly preferred are formulations that target the liver when treating hepatic disorders such as hepatic carcinoma.

The pharmaceutical formulations of the present invention, which can conveniently be presented in unit dosage form, can be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.

The compositions of the present invention can be formulated into any of many possible dosage forms such as, but not limited to, tablets, capsules, gel capsules, liquid syrups, soft gels, suppositories, and enemas. The compositions of the present invention can also be formulated as suspensions in aqueous, non-aqueous, or mixed media. Aqueous suspensions can further contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension can also contain stabilizers.

C. Additional Formulations

i. Emulsions

The compositions of the present invention can be prepared and formulated as emulsions. Emulsions are typically heterogeneous systems of one liquid dispersed in another in the form of droplets usually exceeding 0.1 μm in diameter (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., Volume 1, p. 245; Block in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 2, p. 335; Higuchi et al., in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 301). Emulsions are often biphasic systems comprising two immiscible liquid phases intimately mixed and dispersed with each other. In general, emulsions can be of either the water-in-oil (w/o) or the oil-in-water (o/w) variety. When an aqueous phase is finely divided into and dispersed as minute droplets into a bulk oily phase, the resulting composition is called a water-in-oil (w/o) emulsion. Alternatively, when an oily phase is finely divided into and dispersed as minute droplets into a bulk aqueous phase, the resulting composition is called an oil-in-water (o/w) emulsion. Emulsions can contain additional components in addition to the dispersed phases, and the active drug which can be present as a solution in either aqueous phase, oily phase or itself as a separate phase. Pharmaceutical excipients such as emulsifiers, stabilizers, dyes, and anti-oxidants can also be present in emulsions as needed. Pharmaceutical emulsions can also be multiple emulsions that are comprised of more than two phases such as, for example, in the case of oil-in-water-in-oil (o/w/o) and water-in-oil-in-water (w/o/w) emulsions. Such complex formulations often provide certain advantages that simple binary emulsions do not. Multiple emulsions in which individual oil droplets of an o/w emulsion enclose small water droplets constitute a w/o/w emulsion. Likewise a system of oil droplets enclosed in globules of water stabilized in an oily continuous phase provides an o/w/o emulsion.

Emulsions are characterized by little or no thermodynamic stability. Often, the dispersed or discontinuous phase of the emulsion is well dispersed into the external or continuous phase and maintained in this form through the means of emulsifiers or the viscosity of the formulation. Either of the phases of the emulsion can be a semisolid or a solid, as is the case of emulsion-style ointment bases and creams Other means of stabilizing emulsions entail the use of emulsifiers that can be incorporated into either phase of the emulsion. Emulsifiers can broadly be classified into four categories: synthetic surfactants, naturally occurring emulsifiers, absorption bases, and finely dispersed solids (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).

Synthetic surfactants, also known as surface active agents, have found wide applicability in the formulation of emulsions and have been reviewed in the literature (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), Marcel Dekker, Inc., New York, N.Y., 1988, volume 1, p. 199). Surfactants are typically amphiphilic and comprise a hydrophilic and a hydrophobic portion. The ratio of the hydrophilic to the hydrophobic nature of the surfactant has been termed the hydrophile/lipophile balance (HLB) and is a valuable tool in categorizing and selecting surfactants in the preparation of formulations. Surfactants can be classified into different classes based on the nature of the hydrophilic group: nonionic, anionic, cationic and amphoteric (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y. Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285).

Naturally occurring emulsifiers used in emulsion formulations include lanolin, beeswax, phosphatides, lecithin, and acacia. Absorption bases possess hydrophilic properties such that they can soak up water to form w/o emulsions yet retain their semisolid consistencies, such as anhydrous lanolin and hydrophilic petrolatum. Finely divided solids have also been used as good emulsifiers especially in combination with surfactants and in viscous preparations. These include polar inorganic solids, such as heavy metal hydroxides, nonswelling clays such as bentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidal aluminum silicate and colloidal magnesium aluminum silicate, pigments, and nonpolar solids such as carbon or glyceryl tristearate.

A large variety of non-emulsifying materials are also included in emulsion formulations and contribute to the properties of emulsions. These include fats, oils, waxes, fatty acids, fatty alcohols, fatty esters, humectants, hydrophilic colloids, preservatives and antioxidants (Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).

Hydrophilic colloids or hydrocolloids include naturally occurring gums and synthetic polymers such as polysaccharides (for example, acacia, agar, alginic acid, carrageenan, guar gum, karaya gum, and tragacanth), cellulose derivatives (for example, carboxymethylcellulose and carboxypropylcellulose), and synthetic polymers (for example, carbomers, cellulose ethers, and carboxyvinyl polymers). These disperse or swell in water to form colloidal solutions that stabilize emulsions by forming strong interfacial films around the dispersed-phase droplets and by increasing the viscosity of the external phase.

Since emulsions often contain a number of ingredients such as carbohydrates, proteins, sterols, and phosphatides that can readily support the growth of microbes, these formulations often incorporate preservatives. Commonly used preservatives included in emulsion formulations include methyl paraben, propyl paraben, quaternary ammonium salts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boric acid. Antioxidants are also commonly added to emulsion formulations to prevent deterioration of the formulation. Antioxidants used can be free radical scavengers such as tocopherols, alkyl gallates, butylated hydroxyanisole, butylated hydroxytoluene, or reducing agents such as ascorbic acid and sodium metabisulfite, and antioxidant synergists such as citric acid, tartaric acid, and lecithin.

The application of emulsion formulations via dermatological, oral and parenteral routes and methods for their manufacture have been reviewed in the literature (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Emulsion formulations for oral delivery have been very widely used because of ease of formulation, as well as efficacy from an absorption and bioavailability standpoint (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Mineral-oil base laxatives, oil-soluble vitamins, and high fat nutritive preparations are among the materials that have commonly been administered orally as o/w emulsions.

ii. Microemulsions

In one embodiment of the present invention, the compositions of iRNAs and nucleic acids are formulated as microemulsions. A microemulsion can be defined as a system of water, oil and amphiphile which is a single optically isotropic and thermodynamically stable liquid solution (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245). Typically microemulsions are systems that are prepared by first dispersing an oil in an aqueous surfactant solution and then adding a sufficient amount of a fourth component, generally an intermediate chain-length alcohol to form a transparent system. Therefore, microemulsions have also been described as thermodynamically stable, isotropically clear dispersions of two immiscible liquids that are stabilized by interfacial films of surface-active molecules (Leung and Shah, in: Controlled Release of Drugs: Polymers and Aggregate Systems, Rosoff, M., Ed., 1989, VCH Publishers, New York, pages 185-215). Microemulsions commonly are prepared via a combination of three to five components that include oil, water, surfactant, cosurfactant and electrolyte. Whether the microemulsion is of the water-in-oil (w/o) or an oil-in-water (o/w) type is dependent on the properties of the oil and surfactant used and on the structure and geometric packing of the polar heads and hydrocarbon tails of the surfactant molecules (Schott, in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 271).

The phenomenological approach utilizing phase diagrams has been extensively studied and has yielded a comprehensive knowledge, to one skilled in the art, of how to formulate microemulsions (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335). Compared to conventional emulsions, microemulsions offer the advantage of solubilizing water-insoluble drugs in a formulation of thermodynamically stable droplets that are formed spontaneously.

Surfactants used in the preparation of microemulsions include, but are not limited to, ionic surfactants, non-ionic surfactants, Brij™ 96, polyoxyethylene oleyl ethers, polyglycerol fatty acid esters, tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310), hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500), decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750), decaglycerol sequioleate (SO750), decaglycerol decaoleate (DA0750), alone or in combination with cosurfactants. The cosurfactant, usually a short-chain alcohol such as ethanol, 1-propanol, or 1-butanol, serves to increase the interfacial fluidity by penetrating into the surfactant film and consequently creating a disordered film because of the void space generated among surfactant molecules. Microemulsions can, however, be prepared without the use of cosurfactants and alcohol-free self-emulsifying microemulsion systems are known in the art. The aqueous phase can typically be, but is not limited to, water, an aqueous solution of the drug, glycerol, PEG300, PEG400, polyglycerols, propylene glycols, and derivatives of ethylene glycol. The oil phase can include, but is not limited to, materials such as Captex™ 300, Captex™ 355, Capmul™ MCM, fatty acid esters, medium chain (C8-C12) mono, di, and tri-glycerides, polyoxyethylated glyceryl fatty acid esters, fatty alcohols, polyglycolized glycerides, saturated polyglycolized C8-C10 glycerides, vegetable oils, and silicone oil.

Microemulsions are particularly of interest from the standpoint of drug solubilization and the enhanced absorption of drugs. Lipid based microemulsions (both o/w and w/o) have been proposed to enhance the oral bioavailability of drugs, including peptides (see e.g., U.S. Pat. Nos. 6,191,105; 7,063,860; 7,070,802; 7,157,099; Constantinides et al., Pharmaceutical Research, 1994, 11, 1385-1390; Ritschel, Meth. Find. Exp. Clin. Pharmacol., 1993, 13, 205). Microemulsions afford advantages of improved drug solubilization, protection of drug from enzymatic hydrolysis, possible enhancement of drug absorption due to surfactant-induced alterations in membrane fluidity and permeability, ease of preparation, ease of oral administration over solid dosage forms, improved clinical potency, and decreased toxicity (see e.g., U.S. Pat. Nos. 6,191,105; 7,063,860; 7,070,802; 7,157,099; Constantinides et al., Pharmaceutical Research, 1994, 11, 1385; Ho et al., J. Pharm. Sci., 1996, 85, 138-143). Often microemulsions can form spontaneously when their components are brought together at ambient temperature. This can be particularly advantageous when formulating thermolabile drugs, peptides or iRNAs. Microemulsions have also been effective in the transdermal delivery of active components in both cosmetic and pharmaceutical applications. It is expected that the microemulsion compositions and formulations of the present invention will facilitate the increased systemic absorption of iRNAs and nucleic acids from the gastrointestinal tract, as well as improve the local cellular uptake of iRNAs and nucleic acids.

Microemulsions of the present invention can also contain additional components and additives such as sorbitan monostearate (Grill 3), Labrasol, and penetration enhancers to improve the properties of the formulation and to enhance the absorption of the iRNAs and nucleic acids of the present invention. Penetration enhancers used in the microemulsions of the present invention can be classified as belonging to one of five broad categories—surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Each of these classes has been discussed above.

iii. Microparticles

an RNAi agent of the invention may be incorporated into a particle, e.g., a microparticle. Microparticles can be produced by spray-drying, but may also be produced by other methods including lyophilization, evaporation, fluid bed drying, vacuum drying, or a combination of these techniques.

iv. Penetration Enhancers

In one embodiment, the present invention employs various penetration enhancers to effect the efficient delivery of nucleic acids, particularly iRNAs, to the skin of animals. Most drugs are present in solution in both ionized and nonionized forms. However, usually only lipid soluble or lipophilic drugs readily cross cell membranes. It has been discovered that even non-lipophilic drugs can cross cell membranes if the membrane to be crossed is treated with a penetration enhancer. In addition to aiding the diffusion of non-lipophilic drugs across cell membranes, penetration enhancers also enhance the permeability of lipophilic drugs.

Penetration enhancers can be classified as belonging to one of five broad categories, i.e., surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (see e.g., Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, N.Y., 2002; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Such compounds are well known in the art.

v. Carriers

Certain compositions of the present invention also incorporate carrier compounds in the formulation. As used herein, “carrier compound” or “carrier” can refer to a nucleic acid, or analog thereof, which is inert (i.e., does not possess biological activity per se) but is recognized as a nucleic acid by in vivo processes that reduce the bioavailability of a nucleic acid having biological activity by, for example, degrading the biologically active nucleic acid or promoting its removal from circulation. The coadministration of a nucleic acid and a carrier compound, typically with an excess of the latter substance, can result in a substantial reduction of the amount of nucleic acid recovered in the liver, kidney, or other extracirculatory reservoirs, presumably due to competition between the carrier compound and the nucleic acid for a common receptor. For example, the recovery of a partially phosphorothioate dsRNA in hepatic tissue can be reduced when it is coadministered with polyinosinic acid, dextran sulfate, polycytidic acid or 4-acetamido-4′isothiocyano-stilbene-2,2′-disulfonic acid (Miyao et al., DsRNA Res. Dev., 1995, 5, 115-121; Takakura et al., DsRNA & Nucl. Acid Drug Dev., 1996, 6, 177-183.

vi. Excipients

In contrast to a carrier compound, a “pharmaceutical carrier” or “excipient” is a pharmaceutically acceptable solvent, suspending agent or any other pharmacologically inert vehicle for delivering one or more nucleic acids to an animal. The excipient can be liquid or solid and is selected, with the planned manner of administration in mind, so as to provide for the desired bulk, consistency, etc., when combined with a nucleic acid and the other components of a given pharmaceutical composition. Typical pharmaceutical carriers include, but are not limited to, binding agents (e.g., pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calcium hydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oils, corn starch, polyethylene glycols, sodium benzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodium starch glycolate, etc.); and wetting agents (e.g., sodium lauryl sulphate, etc).

Pharmaceutically acceptable organic or inorganic excipients suitable for non-parenteral administration which do not deleteriously react with nucleic acids can also be used to formulate the compositions of the present invention. Suitable pharmaceutically acceptable carriers include, but are not limited to, water, salt solutions, alcohols, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone; and the like.

Formulations for topical administration of nucleic acids can include sterile and non-sterile aqueous solutions, non-aqueous solutions in common solvents such as alcohols, or solutions of the nucleic acids in liquid or solid oil bases. The solutions can also contain buffers, diluents, and other suitable additives. Pharmaceutically acceptable organic or inorganic excipients suitable for non-parenteral administration which do not deleteriously react with nucleic acids can be used.

Suitable pharmaceutically acceptable excipients include, but are not limited to, water, salt solutions, alcohol, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone, and the like.

vii. Other Components

The compositions of the present invention can additionally contain other adjunct components conventionally found in pharmaceutical compositions, at their art-established usage levels. Thus, for example, the compositions can contain additional, compatible, pharmaceutically-active materials such as, for example, antipruritics, astringents, local anesthetics or anti-inflammatory agents, or can contain additional materials useful in physically formulating various dosage forms of the compositions of the present invention, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents, and stabilizers. However, such materials, when added, should not unduly interfere with the biological activities of the components of the compositions of the present invention. The formulations can be sterilized and, if desired, mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavorings, and/or aromatic substances; and the like which do not deleteriously interact with the nucleic acid(s) of the formulation.

Aqueous suspensions can contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol, and/or dextran. The suspension can also contain stabilizers.

In some embodiments, pharmaceutical compositions featured in the invention include (a) one or more iRNA compounds and (b) one or more agents which function by a non-RNAi mechanism and which are useful in treating a GCK-associated disorder. Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD₅₀ (the dose lethal to 50% of the population) and the ED₅₀ (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD₅₀/ED₅₀. Compounds that exhibit high therapeutic indices are preferred.

The data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of compositions featured herein in the invention lies generally within a range of circulating concentrations that include the ED₅₀ with little or no toxicity. The dosage can vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the methods featured in the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose can be formulated in animal models to achieve a circulating plasma concentration range of the compound or, when appropriate, of the polypeptide product of a target sequence (e.g., achieving a decreased concentration of the polypeptide) that includes the IC₅₀ (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma can be measured, for example, by high performance liquid chromatography.

In addition to their administration, as discussed above, the iRNAs featured in the invention can be administered in combination with other known agents effective in treatment of pathological processes mediated by GCK expression. In any event, the administering physician can adjust the amount and timing of iRNA administration on the basis of results observed using standard measures of efficacy known in the art or described herein.

VI. Methods of the Invention

The present invention also provides methods of using an iRNA of the invention and/or a composition containing an iRNA of the invention to reduce and/or inhibit glucokinase (GCK) expression in a cell. The methods include contacting the cell with a dsRNA of the invention and maintaining the cell for a time sufficient to obtain degradation of the mRNA transcript of a GCK gene, thereby inhibiting expression of the GCK gene in the cell. Reduction in gene expression can be assessed by any methods known in the art. For example, a reduction in the expression of GCK may be determined by determining the mRNA expression level of GCK using methods routine to one of ordinary skill in the art, e.g., northern blotting, qRT-PCR; by determining the protein level of GCK using methods routine to one of ordinary skill in the art, such as western blotting, immunological techniques. A reduction in the expression of GCK may also be assessed indirectly by measuring a decrease in biological activity of GCK.

In the methods of the invention the cell may be contacted in vitro or in vivo, i.e., the cell may be within a subject.

A cell suitable for treatment using the methods of the invention may be any cell that expresses a GCK gene. A cell suitable for use in the methods of the invention may be a mammalian cell, e.g., a primate cell (such as a human cell or a non-human primate cell, e.g., a monkey cell or a chimpanzee cell), a non-primate cell (such as a cow cell, a pig cell, a camel cell, a llama cell, a horse cell, a goat cell, a rabbit cell, a sheep cell, a hamster, a guinea pig cell, a cat cell, a dog cell, a rat cell, a mouse cell, a lion cell, a tiger cell, a bear cell, or a buffalo cell), a bird cell (e.g., a duck cell or a goose cell), or a whale cell. In one embodiment, the cell is a human cell, e.g., a human liver cell.

GCK expression is inhibited in the cell by at least about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or about 100%. In preferred embodiments, GCK expression is inhibited by at least 20%.

The in vivo methods of the invention may include administering to a subject a composition containing an iRNA, where the iRNA includes a nucleotide sequence that is complementary to at least a part of an RNA transcript of the GCK gene of the mammal to be treated. When the organism to be treated is a mammal such as a human, the composition can be administered by any means known in the art including, but not limited to oral, intraperitoneal, or parenteral routes, including intracranial (e.g., intraventricular, intraparenchymal, and intrathecal), intravenous, intramuscular, subcutaneous, transdermal, airway (aerosol), nasal, rectal, and topical (including buccal and sublingual) administration. In certain embodiments, the compositions are administered by intravenous infusion or injection. In certain embodiments, the compositions are administered by subcutaneous injection.

In some embodiments, the administration is via a depot injection. A depot injection may release the iRNA in a consistent way over a prolonged time period. Thus, a depot injection may reduce the frequency of dosing needed to obtain a desired effect, e.g., a desired inhibition of GCK, or a therapeutic or prophylactic effect. A depot injection may also provide more consistent serum concentrations. Depot injections may include subcutaneous injections or intramuscular injections. In preferred embodiments, the depot injection is a subcutaneous injection.

In some embodiments, the administration is via a pump. The pump may be an external pump or a surgically implanted pump. In certain embodiments, the pump is a subcutaneously implanted osmotic pump. In other embodiments, the pump is an infusion pump. An infusion pump may be used for intravenous, subcutaneous, arterial, or epidural infusions. In preferred embodiments, the infusion pump is a subcutaneous infusion pump. In other embodiments, the pump is a surgically implanted pump that delivers the iRNA to the liver.

The mode of administration may be chosen based upon whether local or systemic treatment is desired and based upon the area to be treated. The route and site of administration may be chosen to enhance targeting.

In one aspect, the present invention also provides methods for inhibiting the expression of a GCK gene in a mammal. The methods include administering to the mammal a composition comprising a dsRNA that targets a GCK gene in a cell of the mammal and maintaining the mammal for a time sufficient to obtain degradation of the mRNA transcript of the GCK gene, thereby inhibiting expression of the GCK gene in the cell. Reduction in gene expression can be assessed by any methods known it the art and by methods, e.g. qRT-PCR, described herein. Reduction in protein production can be assessed by any methods known it the art and by methods, e.g. ELISA, described herein. In one embodiment, a puncture liver biopsy sample serves as the tissue material for monitoring the reduction in GCK gene and/or protein expression.

The present invention further provides methods of treatment of a subject in need thereof. The treatment methods of the invention include administering an iRNA of the invention to a subject, e.g., a subject that would benefit from a reduction and/or inhibition of GCK expression, in a therapeutically effective amount of an iRNA targeting a GCK gene or a pharmaceutical composition comprising an iRNA targeting a GCK gene.

In addition, the present invention provides methods of increasing the blood glucose levels, e.g., fasting blood glucose levels, in a subject having a disease or disorder that would benefit from a reduction in GCK expression. The methods include administering an iRNA of the invention to the subject in a therapeutically effective amount of an iRNA targeting a GCK gene or a pharmaceutical composition comprising an iRNA targeting a GCK gene.

Further, the present invention provides methods of decreasing plasma lactate levels in a subject having a disease or disorder that would benefit from a reduction in GCK expression, e.g., a GSD, e.g., type Ia GSD. The methods include administering an iRNA of the invention to the subject in a therapeutically effective amount of an iRNA targeting a GCK gene or a pharmaceutical composition comprising an iRNA targeting a GCK gene.

The present invention also provides methods of decreasing plasma uric acid levels in a subject having a disease or disorder that would benefit from a reduction in GCK expression, e.g., a GSD, e.g., type Ia GSD. The methods include administering an iRNA of the invention to the subject in a therapeutically effective amount of an iRNA targeting a GCK gene or a pharmaceutical composition comprising an iRNA targeting a GCK gene.

The present invention provides methods of decreasing plasma triglyceride levels in a subject having a disease or disorder that would benefit from a reduction in GCK expression, e.g., a GSD, e.g., type Ia GSD. The methods include administering an iRNA of the invention to the subject in a therapeutically effective amount of an iRNA targeting a GCK gene or a pharmaceutical composition comprising an iRNA targeting a GCK gene.

The present invention further provides methods of decreasing total plasma cholesterol levels in a subject having a disease or disorder that would benefit from a reduction in GCK expression, e.g., a GSD, e.g., type Ia GSD. The methods include administering an iRNA of the invention to the subject in a therapeutically effective amount of an iRNA targeting a GCK gene or a pharmaceutical composition comprising an iRNA targeting a GCK gene.

The present invention also provides methods of decreasing hepatomegaly in a subject having a disease or disorder that would benefit from a reduction in GCK expression, e.g., a GSD, e.g., type Ia GSD. The methods include administering an iRNA of the invention to the subject in a therapeutically effective amount of an iRNA targeting a GCK gene or a pharmaceutical composition comprising an iRNA targeting a GCK gene.

An iRNA of the invention may be administered as a “free iRNA.” A free iRNA is administered in the absence of a pharmaceutical composition. The naked iRNA may be in a suitable buffer solution. The buffer solution may comprise acetate, citrate, prolamine, carbonate, or phosphate, or any combination thereof. In one embodiment, the buffer solution is phosphate buffered saline (PBS). The pH and osmolarity of the buffer solution containing the iRNA can be adjusted such that it is suitable for administering to a subject.

Alternatively, an iRNA of the invention may be administered as a pharmaceutical composition, such as a dsRNA liposomal formulation.

Subjects that would benefit from a reduction and/or inhibition of GCK gene expression include those having a glycogen storage disease (GSD), e.g., type Ia GSD. In one embodiment, subjects that would benefit from a reduction and/or inhibition of GCK gene expression are those having one or more signs or symptoms associated with a glycogen storage disease (GSD), e.g., type Ia GSD, including, but not limited to, hypoglycemia, lactic acidosis, hyperuricemia, hyperlipidemia, hepatomegaly, kidney disease as a result of glycogen accumulation, hunger, jitteriness, lethargy, apnea, seizures, diaphoresis, confusion, headaches, dizziness, unusual mood or behavior changes, loss of consciousness, coma, muscle cramps, bleeding diathesis, short stauture, osteoporosis, delayed puberty, gout, renal disease, systemic hypertension, pulmonary hypertension, hepatic adenomas, pancreatitis, anemia, vitamin D deficiency, polycystic ovaries, irregular menstrual cycles, menorrhagia, and eruptive xanthomata.

Treatment of a subject that would benefit from a reduction and/or inhibition of GCK gene expression and normalization of blood glucose levels includes therapeutic treatment (e.g., of a subject suffering from a glycogen storage disease (GSD)) and prophylactic treatment (e.g., of a subject that does not meet the diagnostic criteria of a glycogen storage disease (GSD), or a subject who may be at risk of developing a glycogen storage disease (GSD)).

The invention further provides methods for the use of an iRNA or a pharmaceutical composition thereof, e.g., for treating a subject that would benefit from reduction and/or inhibition of GCK expression, e.g., a subject having a glycogen storage disease (GSD), e.g., type Ia GSD, in combination with other pharmaceuticals and/or other therapeutic methods, e.g., with known pharmaceuticals and/or known therapeutic methods, such as, for example, those which are currently employed for treating these disorders. For example, in certain embodiments, an iRNA targeting GCK is administered to a subject having a GSD in combination with, e.g., antihypertensive agents, such as Diazoxide, somatostatin analogues, such as Octreotide, calcium channel blockers, such as, Nifedipine, Thiazide diuretics, such as chlorothiazide (e.g., in combination with Diazoxide), intravenous infusion of glucagon, parenterally adminsitered dextrose; and/or partial pancreatectomy. In some embodiments, an iRNA targeting GCK is administered to a subject having type Ia GSD in combination with, e.g., a sodium-glucose co-transporter 2 (SGLT2) inhibitor, e.g., Dapagliflozin, Canagliflozin, Ipragliflozin (ASP-1941), Tofogliflozin, Empagliflozin, Sergliflozin etabonate, Remogliflozin etabonate (BHV091009), and Ertugliflozin (PF-04971729/MK-8835).

The iRNA and additional therapeutic agents may be administered at the same time and/or in the same combination, e.g., parenterally, or the additional therapeutic agent can be administered as part of a separate composition or at separate times and/or by another method known in the art or described herein.

In one embodiment, the method includes administering a composition featured herein such that expression of the target GCK gene is decreased, such as for about 1, 2, 3, 4, 5, 6, 7, 8, 12, 16, 18, 24 hours, 28, 32, or about 36 hours. In one embodiment, expression of the target GCK gene is decreased for an extended duration, e.g., at least about two, three, four days or more, e.g., about one week, two weeks, three weeks, or four weeks or longer.

Preferably, the iRNAs useful for the methods and compositions featured herein specifically target RNAs (primary or processed) of the target GCK gene. Compositions and methods for inhibiting the expression of these genes using iRNAs can be prepared and performed as described herein.

Administration of the dsRNA according to the methods of the invention may result in a reduction of the severity, signs, symptoms, and/or markers of such diseases or disorders in a patient with a glycogen storage disease (GSD), e.g., type Ia GSD. By “reduction” in this context is meant a statistically significant decrease in such level. The reduction can be, for example, at least about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.

Efficacy of treatment or prevention of disease can be assessed, for example by measuring disease progression, disease remission, symptom severity, reduction in pain, quality of life, dose of a medication required to sustain a treatment effect, level of a disease marker, or any other measurable parameter appropriate for a given disease being treated or targeted for prevention. It is well within the ability of one skilled in the art to monitor efficacy of treatment or prevention by measuring any one of such parameters, or any combination of parameters. For example, efficacy of treatment of a glycogen storage disease (GSD) may be assessed, for example, by periodic monitoring of, e.g., blood glucose levels. Comparisons of the later readings with the initial readings provide a physician an indication of whether the treatment is effective. It is well within the ability of one skilled in the art to monitor efficacy of treatment or prevention by measuring any one of such parameters, or any combination of parameters. In connection with the administration of an iRNA targeting GCK or pharmaceutical composition thereof, “effective against” a glycogen storage disease (GSD) indicates that administration in a clinically appropriate manner results in a beneficial effect for at least a statistically significant fraction of patients, such as a improvement of symptoms, a cure, a reduction in disease, extension of life, improvement in quality of life, or other effect generally recognized as positive by medical doctors familiar with treating a glycogen storage disease (GSD) and the related causes and effects.

A treatment or preventive effect is evident when there is a statistically significant improvement in one or more parameters of disease status, or by a failure to worsen or to develop symptoms where they would otherwise be anticipated. As an example, a favorable change of at least 10% in a measurable parameter of disease, and preferably at least 20%, 30%, 40%, 50% or more can be indicative of effective treatment. Efficacy for a given iRNA drug or formulation of that drug can also be judged using an experimental animal model for the given disease as known in the art. When using an experimental animal model, efficacy of treatment is evidenced when a statistically significant reduction in a marker or symptom is observed.

Alternatively, the efficacy can be measured by a reduction in the severity of disease as determined by one skilled in the art of diagnosis based on a clinically accepted disease severity grading scale such as those provided above. Any positive change resulting in e.g., lessening of severity of disease measured using the appropriate scale, represents adequate treatment using an iRNA or iRNA formulation as described herein.

Subjects can be administered a therapeutic amount of dsRNA, such as about 0.01 mg/kg to about 200 mg/kg.

The iRNA can be administered by intravenous infusion over a period of time, on a regular basis. In certain embodiments, after an initial treatment regimen, the treatments can be administered on a less frequent basis. Administration of the iRNA can reduce GCK levels, e.g., in a cell, tissue, blood, urine, or other compartment of the patient by at least about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 39, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% or more.

Before administration of a full dose of the iRNA, patients can be administered a smaller dose, such as a 5% infusion reaction, and monitored for adverse effects, such as an allergic reaction. In another example, the patient can be monitored for unwanted immunostimulatory effects, such as increased cytokine (e.g., TNF-alpha or INF-alpha) levels.

Alternatively, the iRNA can be administered subcutaneously, i.e., by subcutaneous injection. One or more injections may be used to deliver the desired daily dose of iRNA to a subject. The injections may be repeated over a period of time. The administration may be repeated on a regular basis. In certain embodiments, after an initial treatment regimen, the treatments can be administered on a less frequent basis. A repeat-dose regimen may include administration of a therapeutic amount of iRNA on a regular basis, such as every other day or to once a year. In certain embodiments, the iRNA is administered about once per month to about once per quarter (i.e., about once every three months).

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the iRNAs and methods featured in the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

EXAMPLES Example 1. iRNA Design, Synthesis, Selection, and In Vitro Evaluation

This Example describes methods for the design, synthesis, selection, and in vitro evaluation of GCK iRNA agents.

Source of Reagents

Where the source of a reagent is not specifically given herein, such reagent can be obtained from any supplier of reagents for molecular biology at a quality/purity standard for application in molecular biology.

Bioinformatics

A set of siRNAs targeting the human GCK, “glucokinase (hexokinase 4)”, (human: NCBI refseqID NM_033507; NCBI GeneID: 2645), as well as toxicology-species GCK orthologs (cynomolgus monkey: XM_005549685; mouse: NM_010292; rat, NM_012565) were designed using custom R and Python scripts. The human NM_033507 REFSEQ mRNA, version 1, has a length of 2442 bases. The rationale and method for the set of siRNA designs is as follows: the predicted efficacy for every potential 19mer iRNA from position 1 through position 2442 (the coding region and 3′ UTR) was determined with a linear model derived the direct measure of mRNA knockdown from more than 20,000 distinct iRNA designs targeting a large number of vertebrate genes. Subsets of the GCK iRNAs were designed with perfect or near-perfect matches between human, cynomolgus and rhesus monkey. A further subset was designed with perfect or near-perfect matches to mouse and rat GCK orthologs. For each strand of the iRNA, a custom Python script was used in a brute force search to measure the number and positions of mismatches between the iRNA and all potential alignments in the target species transcriptome. Extra weight was given to mismatches in the seed region, defined here as positions 2-9 of the antisense oligonucleotide, as well the cleavage site of the iRNA, defined here as positions 10-11 of the antisense oligonucleotide. The relative weight of the mismatches was 2.8; 1.2:1 for seed mismatches, cleavage site, and other positions up through antisense position 19. Mismatches in the first position were ignored. A specificity score was calculated for each strand by assuming the value of each weighted mismatch. Preference was given to iRNAs whose antisense score in human and cynomolgus monkey was >=2.0 and predicted efficacy was >=50% knockdown of the GCK transcript.

Synthesis of GCK Sequences

Synthesis of GCK Single Strands and Duplexes

GCK siRNA sequences were synthesized at 1 umol scale on Mermade 192 synthesizer (BioAutomation) using the solid support mediated phosphoramidite chemistry. The solid support was controlled pore glass (500° A) loaded with custom GalNAc ligand or universal solid support (AM biochemical). Ancillary synthesis reagents, 2′-F and 2′-O-Methyl RNA and deoxy phosphoramidites were obtained from Thermo-Fisher (Milwaukee, Wis.) and Hongene (China). 2′F, 2′-O-Methyl, RNA, DNA and other modified nucleosides were introduced in the sequences using the corresponding phosphoramidites. Synthesis of 3′ GalNAc conjugated single strands was performed on a GalNAc modified CPG support. Custom CPG universal solid support was used for the synthesis of antisense single strands. Coupling time for all phosphoramidites (100 mM in acetonitrile) was 5 min employing 5-Ethylthio-1H-tetrazole (ETT) as activator (0.6 M in acetonitrile). Phosphorothioate linkages were generated using a 50 mM solution of 3-((Dimethylamino-methylidene) amino)-3H-1, 2, 4-dithiazole-3-thione (DDTT, obtained from Chemgenes (Wilmington, Mass., USA) in anhydrous acetonitrile/pyridine (1:1 v/v). Oxidation time was 3 minutes. All sequences were synthesized with final removal of the DMT group (“DMT off”).

Upon completion of the solid phase synthesis, single strands were cleaved from the solid support and deprotected in sealed 96 deep well plates using 200 μL Aqueous Methylamine reagent at 60° C. for 20 minutes. For sequences containing 2′ ribo residues (2′-OH) that are protected with tert-butyl dimethyl silyl (TBDMS) group, a second step deprotection was performed using TEA.3HF (triethylamine trihydro fluoride) reagent. To the methylamine deprotection solution, 200 μL of dimethyl sulfoxide (DMSO) and 300 μl TEA.3HF reagent was added and the solution was incubated for additional 20 min at 60° C. At the end of cleavage and deprotection step, the synthesis plate was allowed to come to room temperature and was precipitated by addition of 1 mL of acetontile:ethanol mixture (9:1). The plates were cooled at −80° C. for 2 hrs and the supernatant decanted carefully with the aid of a multi-channel pipette. The oligonucleotide pellet was re-suspended in 20 mM NaOAc buffer and were desalted using a 5 mL HiTrap size exclusion column (GE Healthcare) on an AKTA Purifier System equipped with an A905 autosampler and a Frac 950 fraction collector. Desalted samples were collected in 96 well plates. Samples from each sequence were analyzed by LC-MS to confirm the identity, UV (260 nm) for quantification and a selected set of samples by IEX chromatography to determine purity.

Annealing of GCK single strands was performed on a Tecan liquid handling robot. Equimolar mixture of sense and antisense single strands were combined and annealed in 96 well plates. After combining the complementary single strands, the 96 well plate was sealed tightly and heated in an oven at 100° C. for 10 minutes and allowed to come slowly to room temperature over a period 2-3 hours. The concentration of each duplex was normalized to 10 uM in 1×PBS and then submitted for in vitro screening assays.

Cell Culture and Transfections for Single Dose and Dose Response Studies

Primary mouse hepatocytes (PMH) (GIBCO) or Primary Cynomolgus monkey hepatocytes (PCH) (Celsis) were transfected by adding 4.9 μl of Opti-MEM plus 0.1 μl of Lipofectamine RNAiMax per well (Invitrogen, Carlsbad Calif. cat #13778-150) to 5 μl of siRNA duplexes per well into a 384-well plate and incubated at room temperature for 15 minutes. Forty μl of William's E Medium (Life Tech) containing about 5×10³ cells were then added to the siRNA mixture. Cells were incubated for 24 hours prior to RNA purification. Single dose experiments were performed at 10 nM and 0.1 nM final duplex concentration.

Total RNA Isolation Using DYNABEADS mRNA Isolation Kit (Invitrogen, Part #: 610-12)

Total RNA was isolated using an automated protocol on a BioTek-EL406 platform using DYNABEADs (Invitrogen, cat #61012). Briefly, 50 μl of Lysis/Binding Buffer and 25 μl of lysis buffer containing 3 μl of magnetic beads were added to the plate with cells. Plates were incubated on an electromagnetic shaker for 10 minutes at room temperature and then magnetic beads were captured and the supernatant was removed. Bead-bound RNA was then washed 2 times with 150 μl Wash Buffer A and once with Wash Buffer B. Beads were then washed with 150 μl Elution Buffer, re-captured and supernatant removed.

cDNA Synthesis Using ABI High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, Calif., Cat #4368813)

Ten μl of a master mix containing 1 μl 10× Buffer, 0.4 μl 25× dNTPs, 1 μl 10× Random primers, 0.5 μl Reverse Transcriptase, 0.5 μl RNase inhibitor and 6.6 μl of H₂O per reaction was added to the RNA isolated as described above. Plates were sealed, mixed, and incubated on an electromagnetic shaker for 10 minutes at room temperature, followed by 2 h 37° C. Plates were then incubated at 80° C. for 8 minutes.

Real Time PCR

Two μl of cDNA was added to a master mix containing 0.5 μl GAPDH TaqMan Probe (Applied Biosystems Cat #4326317E), or 0.5 μl of Custom made Cyno GAPDH Taqman Probe, 0.5 μl GCK mouse probe (Mm00439129 ml) or 0.5 μl cyno probe (Mf02827184 ml) and 5 μl Lightcycler 480 probe master mix (Roche Cat #04887301001) per well in a 384 well plate (Roche cat #04887301001). Real time PCR is done in an Roche Lightcycler Real Time PCR system (Roche) using the ΔΔCt(RQ) assay. Each duplex was tested in two independent transfections.

To calculate relative fold change, real time data are analyzed using the ΔΔCt method and normalized to assays performed with cells transfected with a non-targeting control siRNA, AD-1955

The sense and antisense sequences of AD-1955 are:

SENSE: (SEQ ID NO: 29) cuuAcGcuGAGuAcuucGAdTsdT ANTISENSE: (SEQ ID NO: 30) UCGAAGuACUcAGCGuAAGdTsdT

TABLE 1 Abbreviations of nucleotide monomers used in nucleic acid sequence representation. It will be understood that these monomers, when present in an oligonucleotide, are mutually linked by 5′-3′-phosphodiester bonds. Abbreviation Nucleotide(s) A Adenosine-3′-phosphate Af 2′-fluoroadenosine-3′-phosphate Afs 2′-fluoroadenosine-3′-phosphorothioate As adenosine-3′-phosphorothioate C cytidine-3′-phosphate Cf 2′-fluorocytidine-3′-phosphate Cfs 2′-fluorocytidine-3′-phosphorothioate Cs cytidine-3′-phosphorothioate G guanosine-3′-phosphate Gf 2′-fluoroguanosine-3′-phosphate Gfs 2′-fluoroguanosine-3′-phosphorothioate Gs guanosine-3′-phosphorothioate T 5′-methyluridine-3′-phosphate Tf 2′-fluoro-5-methyluridine-3′-phosphate Tfs 2′-fluoro-5-methyluridine-3′-phosphorothioate Ts 5-methyluridine-3′-phosphorothioate U Uridine-3′-phosphate Uf 2′-fluorouridine-3′-phosphate Ufs 2′-fluorouridine-3′-phosphorothioate Us uridine-3′-phosphorothioate N any nucleotide (G, A, C, T or U) a 2′-O-methyladenosine-3′-phosphate as 2′-O-methyladenosine-3′-phosphorothioate c 2′-O-methylcytidine-3′-phosphate cs 2′-O-methylcytidine-3′-phosphorothioate g 2′-O-methylguanosine-3′-phosphate gs 2′-O-methylguanosine-3′-phosphorothioate t 2′-O-methyl-5-methyluridine-3′-phosphate ts 2′-O-methyl-5-methyluridine-3′-phosphorothioate u 2′-O-methyluridine-3′-phosphate us 2′-O-methyluridine-3′-phosphorothioate s phosphorothioate linkage L96 N-[tris(GalNAc-alkyl)-amidodecanoyl)]-4-hydroxyprolinol Hyp-(GalNAc-alkyl)3 dT 2′-deoxythymidine-3′-phosphate dC 2′-deoxycytidine-3′-phosphate

A detailed list of the unmodified GCK sense and antisense strand sequences is shown in Table 2 and a detailed list of the modified GCK sense and antisense strand sequences is shown in Table 3.

Table 4 shows the results of a single dose screen in primary mouse hepatocytes transfected with the indicated modified siRNAs. Data are expressed as percent of message remaining relative to cells treated with a non-targeting control siRNA.

Table 5 shows the results of a single dose screen in primary Cynomologous hepatocytes transfected with the indicated modified siRNAs. Data are expressed as percent of message remaining relative to cells treated with a non-targeting control siRNA.

TABLE 2 GCK Unmodified Sequences SEQ ID Postion in Antisense Duplex ID Sense ID Sense Sequence (5′-3′) NO: NM_033507.1 ID AD-69366 A-139669 GAGGACCUGAAGAAGGUG 31 _253-273_s A-139670 AUA AD-69368 A-139673 GGACCUGAAGAAGGUGAU 32 _255-275_s A-139674 GAA AD-69411 A-139758 GGACCUGAAGAAGGUGAUA 33 _257-277_s A-139759 AD-69413 A-139762 ACCUGAAGAAGGUGAUGAA 34 _259-279_s A-139763 AD-69367 A-139671 GGUGAUGAGACGGAUGCA 35 _267-287_s A-139672 GAA AD-69369 A-139675 UGAUGAGACGGAUGCAGA 36 _269-289_s A-139676 AGA AD-69412 A-139760 UGAUGAGACGGAUGCAGAA 37 _271-291_s A-139761 AD-69414 A-139764 AUGAGACGGAUGCAGAAGA 38 _273-293_s A-139765 AD-69371 A-139679 ACGGAUGCAGAAGGAGAU 39 _276-296_s A-139680 GGA AD-69416 A-139768 GGAUGCAGAAGGAGAUGGA 40 _280-300_s A-139769 AD-69370 A-139677 ACCCAUGAAGAGGCCAGU 41 _316-336_s A-139678 GUA AD-69415 A-139766 CCAUGAAGAGGCCAGUGUA 42 _320-340_s A-139767 AD-69372 A-139681 CAGUGUGAAGAUGCUGCC 43 _330-350_s A-139682 CAA AD-69417 A-139770 GUGUGAAGAUGCUGCCCAA 44 _334-354_s A-139771 AD-69373 A-139683 GUGAAGGUGGGAGAAGGU 45 _436-456_s A-139684 GAA AD-69418 A-139772 GAAGGUGGGAGAAGGUGA 46 _440-460_s A-139773 AD-69374 A-139685 GAGCGUGAAGACCAAACA 47 _468-488_s A-139686 CCA AD-69419 A-139774 GCGUGAAGACCAAACACCA 48 _472-492_s A-139775 AD-69375 A-139687 GAAGACCAAACACCAGAU 49 _474-494_s A-139688 GUA AD-69420 A-139776 AGACCAAACACCAGAUGUA 50 _478-498_s A-139777 AD-69376 A-139689 GACUUCCUGGACAAGCAU 51 _565-585_s A-139690 CAA AD-69377 A-139691 CUUCCUGGACAAGCAUCA 52 _567-587_s A-139692 GAU AD-69421 A-139778 CUUCCUGGACAAGCAUCAA 53 _569-589_s A-139779 AD-69422 A-139780 UCCUGGACAAGCAUCAGAU 54 _571-591_s A-139781 AD-69378 A-139693 CCUGGACAAGCAUCAGAU 55 _570-590_s A-139694 GAA AD-69379 A-139695 CUGGACAAGCAUCAGAUG 56 _571-591_s A-139696 AAA AD-69423 A-139782 UGGACAAGCAUCAGAUGAA 57 _574_594_s A-139783 AD-69424 A-139784 GGACAAGCAUCAGAUGAAA 58 _575-595_s A-139785 AD-69381 A-139699 AGGCACGAAGACAUCGAU 59 _634-654_s A-139700 AAA AD-69426 A-139788 GCACGAAGACAUCGAUAAA 60 _638-658_s A-139789 AD-69382 A-139701 ACAUCGAUAAGGGCAUCC 61 _644-664_s A-139702 UUA AD-69427 A-139790 AUCGAUAAGGGCAUCCUUA 62 _648-668_s A-139791 AD-69383 A-139703 CUGGACCAAGGGCUUCAA 63 _669-689_s A-139704 GGA AD-69428 A-139792 GGACCAAGGGCUUCAAGGA 64 _673-693_s A-139793 AD-69384 A-139705 GGGCUUCAAGGCCUCAGG 65 _678-698_s A-139706 AGA AD-69429 A-139794 GCUUCAAGGCCUCAGGAGA 66 _682-702_s A-139795 AD-69385 A-139707 CAGGAGCAGAAGGGAACA 67 _692-712_s A-139708 AUA AD-69386 A-139709 GGAGCAGAAGGGAACAAU 68 _694-714_s A-139710 GUA AD-69430 A-139796 GGAGCAGAAGGGAACAAUA 69 _696-716_s A-139797 AD-69387 A-139711 GAGCAGAAGGGAACAAUG 70 _695-715_s A-139712 UCA AD-69431 A-139798 AGCAGAAGGGAACAAUGUA 71 _698-718_s A-139799 AD-69432 A-139800 GCAGAAGGGAACAAUGUCA 72 _699-719_s A-139801 AD-69388 A-139713 GACUUUGAAAUGGAUGUG 73 _751-771_s A-139714 GUA AD-69389 A-139715 CUUUGAAAUGGAUGUGGU 74 _753-773_s A-139716 GGA AD-69433 A-139802 CUUUGAAAUGGAUGUGGUA 75 _755-775_s A-139803 AD-69434 A-139804 UUGAAAUGGAUGUGGUGGA 76 _757-777_s A-139805 AD-69391 A-139719 UGAAAUGGAUGUGGUGGC 77 _756-776_s A-139720 AAU AD-69436 A-139808 AAAUGGAUGUGGUGGCAAU 78 _760-780_s A-139809 AD-69392 A-139721 AUGGAUGUGGUGGCAAUG 79 _760-780_s A-139722 GUA AD-69402 A-139721 AUGGAUGUGGUGGCAAUG 80 _760-780_s A-139741 GUA AD-69393 A-139723 GGAUGUGGUGGCAAUGGU 81 _762-782_s A-139724 GAA AD-69437 A-139810 GGAUGUGGUGGCAAUGGUA 82 _764-784_s A-139811 AD-69438 A-139812 AUGUGGUGGCAAUGGUGAA 83 _766-786_s A-139813 AD-69395 A-139727 GCAAUGGUGAAUGACACG 84 _772-792_s A-139728 GUA AD-69440 A-139816 AAUGGUGAAUGACACGGUA 85 _776-796_s A-139817 AD-69396 A-139729 CAGUGCGAGGUCGGCAUG 86 _826-846_s A-139730 AUA AD-69441 A-139818 GUGCGAGGUCGGCAUGAUA 87 _830-850_s A-139819 AD-69397 A-139731 ACAUGGAGGAGAUGCAGA 88 _872-892_s A-139732 AUA AD-69442 A-139820 AUGGAGGAGAUGCAGAAUA 89 _876-896_s A-139821 AD-69394 A-139725 GAUGCAGAAUGUGGAGCU 90 _882-902_s A-139726 GGU AD-69439 A-139814 UGCAGAAUGUGGAGCUGGU 91 _886-906_s A-139815 AD-69398 A-139733 GUGGACGAGAGCUCUGCA 92 _1000-1020_s A-139734 AAA AD-69443 A-139822 GGACGAGAGCUCUGCAAAA 93 _1004-1024_s A-139823 AD-69380 A-139697 AAGUACAUGGGCGAGCUG 94 _1057-1077_s A-139698 GUA AD-69425 A-139786 GUACAUGGGCGAGCUGGUA 95 _1061-1081_s A-139787 AD-69403 A-139742 AGGCUCGUGGACGAAAAC 96 _1093-1113_s A-139743 CUA AD-69447 A-139830 GCUCGUGGACGAAAACCUA 97 _1097-1117_s A-139831 AD-69404 A-139744 CGUGGACGAAAACCUGCU 98 _1098-1118_s A-139745 CUU AD-69405 A-139746 GUGGACGAAAACCUGCUC 99 _1099-1119_s A-139747 UUA AD-69448 A-139832 UGGACGAAAACCUGCUCUU 100 _1102-1122_s A-139833 AD-69449 A-139834 GGACGAAAACCUGCUCUUA 101 _1103-1123_s A-139835 AD-69406 A-139748 CGCAAGCAGAUCUACAAC 102 _1204-1224_s A-139749 AUA AD-69450 A-139836 CAAGCAGAUCUACAACAUA 103 _1208-1228_s A-139837 AD-69390 A-139717 AGCUGCGAGAUCACCUUC 104 _1468-1488_s A-139718 AUA AD-69435 A-139806 CUGCGAGAUCACCUUCAUA 105 _1472-1492_s A-139807 AD-69408 A-139752 CCAGUCCUGGCCAUUUUC 106 _2049-2069_s A-139753 UUA AD-69452 A-139840 AGUCCUGGCCAUUUUCUUA 107 _2053-2073_s A-139841 AD-69409 A-139754 CACUGAGUGGCUUGUGAU 108 _2170-2190_s A-139755 UCU AD-69453 A-139842 CUGAGUGGCUUGUGAUUCU 109 _2174-2194_s A-139843 AD-69410 A-139756 AAUGUUAAAAGUUUUAAA 110 _2413-2433_s A-139757 CAU AD-69454 A-139844 UGUUAAAAGUUUUAAACAU 111 _2417-2437_s A-139845 AD-69444 A-139824 AGCAGAAGGGAACAACAUA 112 _698-718_s A-139825 AD-69399 A-139735 GGAGCAGAAGGGAACAAC 113 _694-714_s A-139736 AUA AD-69445 A-139826 UCUCCGAGAUGCUAUCAAA 114 _725-745_s A-139827 AD-69400 A-139737 CUUCUCCGAGAUGCUAUC 115 _721-741_s A-139738 AAA AD-69446 A-139828 AGAUGGAUGUGGUGGCAAU 116 _760-780_s A-139829 AD-69401 A-139739 UGAGAUGGAUGUGGUGGC 117 _756-776_s A-139740 AAU AD-69451 A-139838 CUGCGAAAUCACCUUCAUU 118 _1472-1492_s A-139839 AD-69407 A-139750 AACUGCGAAAUCACCUUC 119 _1468-1488_s A-139751 AUU SEQ ID Postion in Duplex ID Antisense Sequence (5′-3′) NO: NM_033507.1 cross_sp AD-69366 UAUCACCUUCUUCAGGUCCU 120 _251-273_as hcmr CCU AD-69368 UUCAUCACCUUCUUCAGGUC 121 _253-275_as hcmr CUC AD-69411 UAUCACCUUCUUCAGGUCC 122 _255-277_as hcmr AD-69413 UUCAUCACCUUCUUCAGGU 123 _257-279_as hcmr AD-69367 UUCUGCAUCCGUCUCAUCAC 124 _265-287_as hcmr CUU AD-69369 UCUUCUGCAUCCGUCUCAUC 125 _267-289_as hcmr ACC AD-69412 UUCUGCAUCCGUCUCAUCA 126 _269-291_as hcmr AD-69414 UCUUCUGCAUCCGUCUCAU 127 _271-293_as hcmr AD-69371 UCCAUCUCCUUCUGCAUCCG 128 _274-296_as hcmr UCU AD-69416 UCCAUCUCCUUCUGCAUCC 129 _278-300_as hcmr AD-69370 UACACUGGCCUCUUCAUGGG 130 _314-336_as hcmr UCU AD-69415 UACACUGGCCUCUUCAUGG 131 _318-340_as hcmr AD-69372 UUGGGCAGCAUCUUCACACU 132 _328-350_as hc GGC AD-69417 UUGGGCAGCAUCUUCACAC 133 _332-354_as hc AD-69373 UUCACCUUCUCCCACCUUCA 134 _434-456_as hc CCA AD-69418 UUCACCUUCUCCCACCUUC 135 _438-460_as hc AD-69374 UGGUGUUUGGUCUUCACGC 136 _466-488_as hc UCCA AD-69419 UGGUGUUUGGUCUUCACGC 137 _470-492_as hc AD-69375 UACAUCUGGUGUUUGGUCU 138 _472-494_as hcmr UCAC AD-69420 UACAUCUGGUGUUUGGUCU 139 _476-498_as hcmr AD-69376 UUGAUGCUUGUCCAGGAAG 140 _563-585_as hcmr UCGG AD-69377 AUCUGAUGCUUGUCCAGGA 141 _565-587_as hcmr AGUC AD-69421 UUGAUGCUUGUCCAGGAAG 142 _567-589_as hcmr AD-69422 AUCUGAUGCUUGUCCAGGA 143 _569-591_as hcmr AD-69378 UUCAUCUGAUGCUUGUCCAG 144 _568-590_as hcmr GAA AD-69379 UUUCAUCUGAUGCUUGUCCA 145 _569-591_as hcmr GGA AD-69423 UUCAUCUGAUGCUUGUCCA 146 _572-594_as hcmr AD-69424 UUUCAUCUGAUGCUUGUCC 147 _573-595_as hcmr AD-69381 UUUAUCGAUGUCUUCGUGCC 148 _632-654_as hc UCA AD-69426 UUUAUCGAUGUCUUCGUGC 149 _636-658_as hc AD-69382 UAAGGAUGCCCUUAUCGAU 150 _642-664_as hc GUCU AD-69427 UAAGGAUGCCCUUAUCGAU 151 _646-668_as hc AD-69383 UCCUUGAAGCCCUUGGUCCA 152 _667-689_as hcmr GUU AD-69428 UCCUUGAAGCCCUUGGUCC 153 _671-693_as hcmr AD-69384 UCUCCUGAGGCCUUGAAGCC 154 _676-698_as hc CUU AD-69429 UCUCCUGAGGCCUUGAAGC 155 _680-702_as hc AD-69385 UAUUGUUCCCUUCUGCUCCU 156 _690-712_as hc GAG AD-69386 UACAUUGUUCCCUUCUGCUC 157 _692-714_as hc CUG AD-69430 UAUUGUUCCCUUCUGCUCC 158 _694-716_as hc AD-69387 UGACAUUGUUCCCUUCUGCU 159 _693-715_as hc CCU AD-69431 UACAUUGUUCCCUUCUGCU 160 _696-718_as hc AD-69432 UGACAUUGUUCCCUUCUGC 161 _697-719_as hc AD-69388 UACCACAUCCAUUUCAAAGU 162 _749-771_as hcmr CCC AD-69389 UCCACCACAUCCAUUUCAAA 163 _751-773_as hcmr GUC AD-69433 UACCACAUCCAUUUCAAAG 164 _753-775_as hcmr AD-69434 UCCACCACAUCCAUUUCAA 165 _755-777_as hcmr AD-69391 AUUGCCACCACAUCCAUUUC 166 _754-776_as hcmr AAA AD-69436 AUUGCCACCACAUCCAUUU 167 _758-780_as hcmr AD-69392 UACCAUUGCCACCACAUCCA 168 _758-780_as hcmr UUU AD-69402 UACCAUUGCCACCACAUCCA 169 _758-780_as mr UCU AD-69393 UUCACCAUUGCCACCACAUC 170 _760-782_as hcmr CAU AD-69437 UACCAUUGCCACCACAUCC 171 _762-784_as hcmr AD-69438 UUCACCAUUGCCACCACAU 172 _764-786_as hcmr AD-69395 UACCGUGUCAUUCACCAUUG 173 _770-792_as hc CCA AD-69440 UACCGUGUCAUUCACCAUU 174 _774-796_as hc AD-69396 UAUCAUGCCGACCUCGCACU 175 _824-846_as hcmr GAU AD-69441 UAUCAUGCCGACCUCGCAC 176 _828-850_as hcmr AD-69397 UAUUCUGCAUCUCCUCCAUG 177 _870-892_as hc UAG AD-69442 UAUUCUGCAUCUCCUCCAU 178 _874-896_as hc AD-69394 ACCAGCUCCACAUUCUGCAU 179 _880-902_as hcmr CUC AD-69439 ACCAGCUCCACAUUCUGCA 180 _884-906_as hcmr AD-69398 UUUUGCAGAGCUCUCGUCCA 181 _998-1020_as hc CCA AD-69443 UUUUGCAGAGCUCUCGUCC 182 _1002-1024_as hc AD-69380 UACCAGCUCGCCCAUGUACU 183 _1055-1077_as hcmr UGC AD-69425 UACCAGCUCGCCCAUGUAC 184 _1059-1081_as hcmr AD-69403 UAGGUUUUCGUCCACGAGCC 185 _1091-1113_as hc UGA AD-69447 UAGGUUUUCGUCCACGAGC 186 _1095-1117_as hc AD-69404 AAGAGCAGGUUUUCGUCCAC 187 _1096-1118_as hc GAG AD-69405 UAAGAGCAGGUUUUCGUCC 188 _1097-1119_as hc ACGA AD-69448 AAGAGCAGGUUUUCGUCCA 189 _1100-1122_as hc AD-69449 UAAGAGCAGGUUUUCGUCC 190 _1101-1123_as hc AD-69406 UAUGUUGUAGAUCUGCUUG 191 _1202-1224_as hc CGGU AD-69450 UAUGUUGUAGAUCUGCUUG 192 _1206-1228_as hc AD-69390 UAUGAAGGUGAUCUCGCAG 193 _1466-1488_as hcmr CUGG AD-69435 UAUGAAGGUGAUCUCGCAG 194 _1470-1492_as hcmr AD-69408 UAAGAAAAUGGCCAGGACU 195 _2047-2069_as hc GGGU AD-69452 UAAGAAAAUGGCCAGGACU 196 _2051-2073_as hc AD-69409 AGAAUCACAAGCCACUCAGU 197 _2168-2190_as hc GAU AD-69453 AGAAUCACAAGCCACUCAG 198 _2172-2194_as hc AD-69410 AUGUUUAAAACUUUUAACA 199 _2411-2433_as hc UUUU AD-69454 AUGUUUAAAACUUUUAACA 200 _2415-2437_as hc AD-69444 UAUGUUGUUCCCUUCUGCU 201 _696-718_as mr AD-69399 UAUGUUGUUCCCUUCUGCUC 202 _692-714_as mr CGG AD-69445 UUUGAUAGCAUCUCGGAGA 203 _723-745_as mr AD-69400 UUUGAUAGCAUCUCGGAGA 204 _719-741_as mr AGUC AD-69446 AUUGCCACCACAUCCAUCU 205 _758-780_as mr AD-69401 AUUGCCACCACAUCCAUCUC 206 _754-776_as mr AAA AD-69451 AAUGAAGGUGAUUUCGCAG 207 _1470-1492_as mr AD-69407 AAUGAAGGUGAUUUCGCAG 208 _1466-1488_as mr UUGG

TABLE 3 GCK Modified Sequences SEQ Anti- SEQ SEQ Duplex ID sense ID ID ID Sense ID Sense sequence (5′-3′) NO: ID Antisense sequence (5′-3′) NO: mRNA sequence NO: AD-69366 A-139669 gsasggacCfuGfAfAfgaaggug 209 A-139670 usAfsucaCfcUfUfcuucAfgG 298 AGGAGGACCUGAAGAA 387 auaL96 fuccucscsu GGUGAUA AD-69368 A-139673 gsgsaccuGfaAfGfAfaggugau 210 A-139674 usUfscauCfaCfCfuucuUfcAf 299 GAGGACCUGAAGAAGG 388 gaaL96 gguccsusc UGAUGAA AD-69411 A-139758 GGACCUGAAGAAGGUG 211 A-139759 UAUCACCUUCUUCAGG 300 GGACCUGAAGAAGGUG 389 AUAdTdT UCCdTdT AUA AD-69413 A-139762 ACCUGAAGAAGGUGAU 212 A-139763 UUCAUCACCUUCUUCA 301 ACCUGAAGAAGGUGAU 390 GAAdTdT GGUdTdT GAA AD-69367 A-139671 gsgsugauGfaGfAfCfggaugca 213 A-139672 usUfscugCfaUfCfcgucUfcAf 302 AAGGUGAUGAGACGGA 391 gaaL96 ucaccsusu UGCAGAA AD-69369 A-139675 usgsaugaGfaCfGfGfaugcaga 214 A-139676 usCfsuucUfgCfAfuccgUfcU 303 GGUGAUGAGACGGAUG 392 agaL96 fcaucascsc CAGAAGA AD-69412 A-139760 UGAUGAGACGGAUGCA 215 A-139761 UUCUGCAUCCGUCUCA 304 UGAUGAGACGGAUGCA 393 GAAdTdT UCAdTdT GAA AD-69414 A-139764 AUGAGACGGAUGCAGA 216 A-139765 UCUUCUGCAUCCGUCU 305 AUGAGACGGAUGCAGA 394 AGAdTdT CAUdTdT AGA AD-69371 A-139679 ascsggauGfcAfGfAfaggagau 217 A-139680 usCfscauCfuCfCfuucuGfcAf 306 AGACGGAUGCAGAAGG 395 ggaL96 uccguscsu AGAUGGA AD-69416 A-139768 GGAUGCAGAAGGAGAU 218 A-139769 UCCAUCUCCUUCUGCA 307 GGAUGCAGAAGGAGAU 396 GGAdTdT UCCdTdT GGA AD-69370 A-139677 ascsccauGfaAfGfAfggccagu 219 A-139678 usAfscacUfgGfCfcucuUfcAf 308 AGACCCAUGAAGAGGC 397 guaL96 uggguscsu CAGUGUA AD-69415 A-139766 CCAUGAAGAGGCCAGU 220 A-139767 UACACUGGCCUCUUCA 309 CCAUGAAGAGGCCAGU 398 GUAdTdT UGGdTdT GUA AD-69372 A-139681 csasguguGfaAfGfAfugcugcc 221 A-139682 usUfsgggCfaGfCfaucuUfcA 310 GCCAGUGUGAAGAUGC 399 caaL96 fcacugsgsc UGCCCAA AD-69417 A-139770 GUGUGAAGAUGCUGCC 222 A-139771 UUGGGCAGCAUCUUCA 311 GUGUGAAGAUGCUGCC 400 CAAdTdT CACdTdT CAA AD-69373 A-139683 gsusgaagGfuGfGfGfagaaggu 223 A-139684 usUfscacCfuUfCfucccAfcCf 312 UGGUGAAGGUGGGAGA 401 gaaL96 uucacscsa AGGUGAA AD-69418 A-139772 GAAGGUGGGAGAAGGU 224 A-139773 UUCACCUUCUCCCACC 313 GAAGGUGGGAGAAGGU 402 GAAdTdT UUCdTdT GAA AD-69374 A-139685 gsasgcguGfaAfGfAfccaaaca 225 A-139686 usGfsgugUfuUfGfgucuUfcA 314 UGGAGCGUGAAGACCA 403 ccaL96 fcgcucscsa AACACCA AD-69419 A-139774 GCGUGAAGACCAAACA 226 A-139775 UGGUGUUUGGUCUUCA 315 GCGUGAAGACCAAACA 404 CCAdTdT CGCdTdT CCA AD-69375 A-139687 gsasagacCfaAfAfCfaccagau 227 A-139688 usAfscauCfuGfGfuguuUfgG 316 GUGAAGACCAAACACC 405 guaL96 fucuucsasc AGAUGUA AD-69420 A-139776 AGACCAAACACCAGAU 228 A-139777 UACAUCUGGUGUUUGG 317 AGACCAAACACCAGAU 406 GUAdTdT UCUdTdT GUA AD-69376 A-139689 gsascuucCfuGfGfAfcaagcau 229 A-139690 usUfsgauGfcUfUfguccAfgG 318 CCGACUUCCUGGACAA 407 caaL96 faagucsgsg GCAUCAA AD-69377 A-139691 csusuccuGfgAfCfAfagcauca 230 A-139692 asUfscugAfuGfCfuuguCfcA 319 GACUUCCUGGACAAGC 408 gauL96 fggaagsusc AUCAGAU AD-69421 A-139778 CUUCCUGGACAAGCAU 231 A-139779 UUGAUGCUUGUCCAGG 320 CUUCCUGGACAAGCAU 409 CAAdTdT AAGdTdT CAA AD-69422 A-139780 UCCUGGACAAGCAUCA 232 A-139781 AUCUGAUGCUUGUCCA 321 UCCUGGACAAGCAUCA 410 GAUdTdT GGAdTdT GAU AD-69378 A-139693 cscsuggaCfaAfGfCfaucagau 233 A-139694 usUfscauCfuGfAfugcuUfgU 322 UUCCUGGACAAGCAUC 411 gaaL96 fccaggsasa AGAUGAA AD-69379 A-139695 csusggacAfaGfCfAfucagaug 234 A-139696 usUfsucaUfcUfGfaugcUfuG 323 UCCUGGACAAGCAUCA 412 aaaL96 fuccagsgsa GAUGAAA AD-69423 A-139782 UGGACAAGCAUCAGAU 235 A-139783 UUCAUCUGAUGCUUGU 324 UGGACAAGCAUCAGAU 413 GAAdTdT CCAdTdT GAA AD-69424 A-139784 GGACAAGCAUCAGAUG 236 A-139785 UUUCAUCUGAUGCUUG 325 GGACAAGCAUCAGAUG 414 AAAdTdT UCCdTdT AAA AD-69381 A-139699 asgsgcacGfaAfGfAfcaucgau 237 A-139700 usUfsuauCfgAfUfgucuUfcG 326 UGAGGCACGAAGACAU 415 aaaL96 fugccuscsa CGAUAAA AD-69426 A-139788 GCACGAAGACAUCGAU 238 A-139789 UUUAUCGAUGUCUUCG 327 GCACGAAGACAUCGAU 416 AAAdTdT UGCdTdT AAA AD-69382 A-139701 ascsaucgAfuAfAfGfggcaucc 239 A-139702 usAfsaggAfuGfCfccuuAfuC 328 AGACAUCGAUAAGGGC 417 uuaL96 fgauguscsu AUCCUUA AD-69427 A-139790 AUCGAUAAGGGCAUCC 240 A-139791 UAAGGAUGCCCUUAUC 329 AUCGAUAAGGGCAUCC 418 UUAdTdT GAUdTdT UUA AD-69383 A-139703 csusggacCfaAfGfGfgcuucaa 241 A-139704 usCfscuuGfaAfGfcccuUfgG 330 AACUGGACCAAGGGCU 419 ggaL96 fuccagsusu UCAAGGA AD-69428 A-139792 GGACCAAGGGCUUCAA 242 A-139793 UCCUUGAAGCCCUUGG 331 GGACCAAGGGCUUCAA 420 GGAdTdT UCCdTdT GGA AD-69384 A-139705 gsgsgcuuCfaAfGfGfccucagg 243 A-139706 usCfsuccUfgAfGfgccuUfgA 332 AAGGGCUUCAAGGCCU 421 agaL96 fagcccsusu CAGGAGA AD-69429 A-139794 GCUUCAAGGCCUCAGG 244 A-139795 UCUCCUGAGGCCUUGA 333 GCUUCAAGGCCUCAGG 422 AGAdTdT AGCdTdT AGA AD-69385 A-139707 csasggagCfaGfAfAfgggaaca 245 A-139708 usAfsuugUfuCfCfcuucUfgC 334 CUCAGGAGCAGAAGGG 423 auaL96 fuccugsasg AACAAUA AD-69386 A-139709 gsgsagcaGfaAfGfGfgaacaau 246 A-139710 usAfscauUfgUfUfcccuUfcU 335 CAGGAGCAGAAGGGAA 424 guaL96 fgcuccsusg CAAUGUA AD-69430 A-139796 GGAGCAGAAGGGAACA 247 A-139797 UAUUGUUCCCUUCUGC 336 GGAGCAGAAGGGAACA 425 AUAdTdT UCCdTdT AUA AD-69387 A-139711 gsasgcagAfaGfGfGfaacaaug 248 A-139712 usGfsacaUfuGfUfucccUfuCf 337 AGGAGCAGAAGGGAAC 426 ucaL96 ugcucscsu AAUGUCA AD-69431 A-139798 AGCAGAAGGGAACAAU 249 A-139799 UACAUUGUUCCCUUCU 338 AGCAGAAGGGAACAAU 427 GUAdTdT GCUdTdT GUA AD-69432 A-139800 GCAGAAGGGAACAAUG 250 A-139801 UGACAUUGUUCCCUUC 339 GCAGAAGGGAACAAUG 428 UCAdTdT UGCdTdT UCA AD-69388 A-139713 gsascuuuGfaAfAfUfggaugug 251 A-139714 usAfsccaCfaUfCfcauuUfcAf 340 GGGACUUUGAAAUGGA 429 guaL96 aagucscsc UGUGGUA AD-69389 A-139715 csusuugaAfaUfGfGfauguggu 252 A-139716 usCfscacCfaCfAfuccaUfuUf 341 GACUUUGAAAUGGAUG 430 ggaL96 caaagsusc UGGUGGA AD-69433 A-139802 CUUUGAAAUGGAUGUG 253 A-139803 UACCACAUCCAUUUCA 342 CUUUGAAAUGGAUGUG 431 GUAdTdT AAGdTdT GUA AD-69434 A-139804 UUGAAAUGGAUGUGGU 254 A-139805 UCCACCACAUCCAUUU 343 UUGAAAUGGAUGUGGU 432 GGAdTdT CAAdTdT GGA AD-69391 A-139719 usgsaaauGfgAfUfGfugguggc 255 A-139720 asUfsugcCfaCfCfacauCfcAf 344 UUUGAAAUGGAUGUGG 433 aauL96 uuucasasa UGGCAAU AD-69436 A-139808 AAAUGGAUGUGGUGGC 256 A-139809 AUUGCCACCACAUCCA 345 AAAUGGAUGUGGUGGC 434 AAUdTdT UUUdTdT AAU AD-69392 A-139721 asusggauGfuGfGfUfggcaaug 257 A-139722 usAfsccaUfuGfCfcaccAfcAf 346 AAAUGGAUGUGGUGGC 435 guaL96 uccaususu AAUGGUA AD-69402 A-139721 asusggauGfuGfGfUfggcaaug 258 A-139741 usAfsccaUfuGfCfcaccAfcAf 347 AGAUGGAUGUGGUGGC 436 guaL96 uccauscsu AAUGGUA AD-69393 A-139723 gsgsauguGfgUfGfGfcaauggu 259 A-139724 usUfscacCfaUfUfgccaCfcAf 348 AUGGAUGUGGUGGCAA 437 gaaL96 cauccsasu UGGUGAA AD-69437 A-139810 GGAUGUGGUGGCAAUG 260 A-139811 UACCAUUGCCACCACA 349 GGAUGUGGUGGCAAUG 438 GUAdTdT UCCdTdT GUA AD-69438 A-139812 AUGUGGUGGCAAUGGU 261 A-139813 UUCACCAUUGCCACCA 350 AUGUGGUGGCAAUGGU 439 GAAdTdT CAUdTdT GAA AD-69395 A-139727 gscsaaugGfuGfAfAfugacacg 262 A-139728 usAfsccgUfgUfCfauucAfcCf 351 UGGCAAUGGUGAAUGA 440 guaL96 auugcscsa CACGGUA AD-69440 A-139816 AAUGGUGAAUGACACG 263 A-139817 UACCGUGUCAUUCACC 352 AAUGGUGAAUGACACG 441 GUAdTdT AUUdTdT GUA AD-69396 A-139729 csasgugcGfaGfGfUfcggcaug 264 A-139730 usAfsucaUfgCfCfgaccUfcGf 353 AUCAGUGCGAGGUCGG 442 auaL96 cacugsasu CAUGAUA AD-69441 A-139818 GUGCGAGGUCGGCAUG 265 A-139819 UAUCAUGCCGACCUCG 354 GUGCGAGGUCGGCAUG 443 AUAdTdT CACdTdT AUA AD-69397 A-139731 ascsauggAfgGfAfGfaugcaga 266 A-139732 usAfsuucUfgCfAfucucCfuC 355 CUACAUGGAGGAGAUG 444 auaL96 fcaugusasg CAGAAUA AD-69442 A-139820 AUGGAGGAGAUGCAGA 267 A-139821 UAUUCUGCAUCUCCUC 356 AUGGAGGAGAUGCAGA 445 AUAdTdT CAUdTdT AUA AD-69394 A-139725 gsasugcaGfaAfUfGfuggagcu 268 A-139726 asCfscagCfuCfCfacauUfcUf 357 GAGAUGCAGAAUGUGG 446 gguL96 gcaucsusc AGCUGGU AD-69439 A-139814 UGCAGAAUGUGGAGCU 269 A-139815 ACCAGCUCCACAUUCU 358 UGCAGAAUGUGGAGCU 447 GGUdTdT GCAdTdT GGU AD-69398 A-139733 gsusggacGfaGfAfGfcucugca 270 A-139734 usUfsuugCfaGfAfgcucUfcG 359 UGGUGGACGAGAGCUC 448 aaaL96 fuccacscsa UGCAAAA AD-69443 A-139822 GGACGAGAGCUCUGCA 271 A-139823 UUUUGCAGAGCUCUCG 360 GGACGAGAGCUCUGCA 449 AAAdTdT UCCdTdT AAA AD-69380 A-139697 asasguacAfuGfGfGfcgagcug 272 A-139698 usAfsccaGfcUfCfgcccAfuGf 361 GCAAGUACAUGGGCGA 450 guaL96 uacuusgsc GCUGGUA AD-69425 A-139786 GUACAUGGGCGAGCUG 273 A-139787 UACCAGCUCGCCCAUG 362 GUACAUGGGCGAGCUG 451 GUAdTdT UACdTdT GUA AD-69403 A-139742 asgsgcucGfuGfGfAfcgaaaac 274 A-139743 usAfsgguUfuUfCfguccAfcG 363 UCAGGCUCGUGGACGA 452 cuaL96 fagccusgsa AAACCUA AD-69447 A-139830 GCUCGUGGACGAAAAC 275 A-139831 UAGGUUUUCGUCCACG 364 GCUCGUGGACGAAAAC 453 CUAdTdT AGCdTdT CUA AD-69404 A-139744 csgsuggaCfgAfAfAfaccugcu 276 A-139745 asAfsgagCfaGfGfuuuuCfgU 365 CUCGUGGACGAAAACC 454 cuuL96 fccacgsasg UGCUCUU AD-69405 A-139746 gsusggacGfaAfAfAfccugcuc 277 A-139747 usAfsagaGfcAfGfguuuUfcG 366 UCGUGGACGAAAACCU 455 uuaL96 fuccacsgsa GCUCUUA AD-69448 A-139832 UGGACGAAAACCUGCU 278 A-139833 AAGAGCAGGUUUUCGU 367 UGGACGAAAACCUGCU 456 CUUdTdT CCAdTdT CUU AD-69449 A-139834 GGACGAAAACCUGCUC 279 A-139835 UAAGAGCAGGUUUUCG 368 GGACGAAAACCUGCUC 457 UUAdTdT UCCdTdT UUA AD-69406 A-139748 csgscaagCfaGfAfUfcuacaaca 280 A-139749 usAfsuguUfgUfAfgaucUfgC 369 ACCGCAAGCAGAUCUA 458 uaL96 fuugcgsgsu CAACAUA AD-69450 A-139836 CAAGCAGAUCUACAAC 281 A-139837 UAUGUUGUAGAUCUGC 370 CAAGCAGAUCUACAAC 459 AUAdTdT UUGdTdT AUA AD-69390 A-139717 asgscugcGfaGfAfUfcaccuuc 282 A-139718 usAfsugaAfgGfUfgaucUfcG 371 CCAGCUGCGAGAUCAC 460 auaL96 fcagcusgsg CUUCAUA AD-69435 A-139806 CUGCGAGAUCACCUUC 283 A-139807 UAUGAAGGUGAUCUCG 372 CUGCGAGAUCACCUUC 461 AUAdTdT CAGdTdT AUA AD-69408 A-139752 cscsagucCfuGfGfCfcauuuuc 284 A-139753 usAfsagaAfaAfUfggccAfgG 373 ACCCAGUCCUGGCCAU 462 uuaL96 facuggsgsu UUUCUUA AD-69452 A-139840 AGUCCUGGCCAUUUUC 285 A-139841 UAAGAAAAUGGCCAGG 374 AGUCCUGGCCAUUUUC 463 UUAdTdT ACUdTdT UUA AD-69409 A-139754 csascugaGfuGfGfCfuugugau 286 A-139755 asGfsaauCfaCfAfagccAfcUf 375 AUCACUGAGUGGCUUG 464 ucuL96 cagugsasu UGAUUCU AD-69453 A-139842 CUGAGUGGCUUGUGAU 287 A-139843 AGAAUCACAAGCCACU 376 CUGAGUGGCUUGUGAU 465 UCUdTdT CAGdTdT UCU AD-69410 A-139756 asasuguuAfaAfAfGfuuuuaaa 288 A-139757 asUfsguuUfaAfAfacuuUfuA 377 AAAAUGUUAAAAGUUU 466 cauL96 facauususu UAAACAU AD-69454 A-139844 UGUUAAAAGUUUUAAA 289 A-139845 AUGUUUAAAACUUUUA 378 UGUUAAAAGUUUUAAA 467 CAUdTdT ACAdTdT CAU AD-69444 A-139824 AGCAGAAGGGAACAAC 290 A-139825 UAUGUUGUUCCCUUCU 379 AGCAGAAGGGAACAAC 468 AUAdTdT GCUdTdT AUA AD-69399 A-139735 gsgsagcaGfaAfGfGfgaacaac 291 A-139736 usAfsuguUfgUfUfcccuUfcU 380 CCGGAGCAGAAGGGAA 469 auaL96 fgcuccsgsg CAACAUA AD-69445 A-139826 UCUCCGAGAUGCUAUC 292 A-139827 UUUGAUAGCAUCUCGG 381 UCUCCGAGAUGCUAUC 470 AAAdTdT AGAdTdT AAA AD-69400 A-139737 csusucucCfgAfGfAfugcuauc 293 A-139738 usUfsugaUfaGfCfaucuCfgG 382 GACUUCUCCGAGAUGC 471 aaaL96 fagaagsusc UAUCAAA AD-69446 A-139828 AGAUGGAUGUGGUGGC 294 A-139829 AUUGCCACCACAUCCA 383 AGAUGGAUGUGGUGGC 472 AAUdTdT UCUdTdT AAU AD-69401 A-139739 usgsagauGfgAfUfGfuggugg 295 A-139740 asUfsugcCfaCfCfacauCfcAf 384 UUUGAGAUGGAUGUGG 473 caauL96 ucucasasa UGGCAAU AD-69451 A-139838 CUGCGAAAUCACCUUC 296 A-139839 AAUGAAGGUGAUUUCG 385 CUGCGAAAUCACCUUC 474 AUUdTdT CAGdTdT AUU AD-69407 A-139750 asascugcGfaAfAfUfcaccuuc 297 A-139751 asAfsugaAfgGfUfgauuUfcG 386 CCAACUGCGAAAUCAC 475 auuL96 fcaguusgsg CUUCAUU

TABLE 4 GCK Single Dose Screen in Primary Mouse Hepatocytes Primary Mouse Hepatocytes Duplex Name 10 nM Avg 10 nM SD 0.1 nM Avg 0.1 nM SD AD-69366 82.2 54.1 70.2 12.8 AD-69368 47.8 5.3 91.7 3.6 AD-69411 17.6 4.8 27.9 2.8 AD-69413 15.9 5.1 43.7 4.8 AD-69367 58.2 11.0 92.0 10.5 AD-69369 60.3 10.6 88.0 6.5 AD-69412 24.1 5.3 71.7 15.6 AD-69414 52.5 7.7 82.7 7.2 AD-69371 77.5 9.7 89.1 12.3 AD-69416 11.8 2.4 25.1 4.0 AD-69370 99.5 10.1 96.9 5.1 AD-69415 90.3 9.3 91.3 15.2 AD-69372 27.9 2.2 49.8 10.2 AD-69417 29.9 2.1 63.6 12.6 AD-69373 96.7 17.1 104.7 48.8 AD-69418 86.2 34.0 62.0 9.9 AD-69374 33.7 4.8 71.7 7.8 AD-69419 17.1 3.2 44.8 4.4 AD-69375 72.0 8.3 104.5 5.3 AD-69420 20.3 2.0 46.6 7.2 AD-69376 22.1 2.3 59.0 9.7 AD-69377 23.0 2.0 59.2 6.6 AD-69421 16.1 2.1 29.9 3.5 AD-69422 15.2 1.2 38.9 2.4 AD-69378 24.8 2.1 70.1 4.2 AD-69379 11.2 0.9 18.0 3.2 AD-69423 11.7 1.3 41.1 3.6 AD-69424 9.5 1.6 13.9 3.5 AD-69381 73.6 13.3 99.9 36.4 AD-69426 84.5 6.3 76.5 6.1 AD-69382 91.9 5.6 91.6 3.3 AD-69427 97.6 2.5 88.8 4.5 AD-69383 95.9 3.2 100.5 9.3 AD-69428 32.7 4.3 66.1 5.0 AD-69384 88.3 9.8 97.9 8.6 AD-69429 90.7 10.6 95.9 5.7 AD-69385 29.2 4.2 45.1 4.4 AD-69386 92.5 9.0 97.8 9.8 AD-69430 21.2 1.4 31.8 2.1 AD-69387 69.7 9.0 77.8 7.1 AD-69432 69.5 3.2 79.6 13.6 AD-69388 66.9 11.3 84.7 11.9 AD-69389 76.3 18.3 86.9 6.0 AD-69433 70.0 2.6 76.4 5.0 AD-69434 97.6 6.6 71.6 9.5 AD-69391 21.7 5.1 59.8 5.6 AD-69436 19.9 1.9 54.3 14.6 AD-69392 39.2 4.8 81.5 12.7 AD-69402 32.2 5.1 62.9 12.4 AD-69393 34.9 5.4 78.5 13.0 AD-69437 14.0 2.5 27.3 4.6 AD-69438 11.7 0.7 28.4 2.5 AD-69395 28.2 6.8 55.4 12.2 AD-69440 19.4 2.8 34.7 7.3 AD-69396 29.7 3.0 57.2 9.8 AD-69441 23.8 4.1 44.6 7.4 AD-69397 39.5 11.8 67.9 11.6 AD-69442 29.3 2.4 40.1 8.4 AD-69394 39.4 4.2 79.5 3.2 AD-69439 37.6 3.2 65.9 3.0 AD-69398 92.2 9.6 111.3 16.2 AD-69443 115.1 14.5 97.4 5.1 AD-69380 23.6 3.2 65.0 11.2 AD-69425 11.9 1.2 29.4 6.5 AD-69403 64.2 13.0 77.0 13.9 AD-69447 87.4 7.9 84.3 11.7 AD-69404 67.2 10.6 83.9 16.6 AD-69405 69.0 10.9 86.1 23.6 AD-69448 107.8 6.4 120.4 36.9 AD-69449 129.6 37.4 120.2 6.4 AD-69406 63.8 4.7 91.7 10.8 AD-69450 84.5 12.4 109.5 5.3 AD-69390 16.9 4.2 38.9 11.6 AD-69435 9.0 1.5 20.9 3.5 AD-69408 67.3 4.7 83.6 16.3 AD-69452 92.6 10.4 111.8 5.1 AD-69409 74.8 4.8 79.7 12.3 AD-69453 87.2 1.4 93.7 3.0 AD-69410 71.2 4.9 90.2 9.4 AD-69454 90.0 10.9 86.0 7.8 AD-69399 24.4 2.5 45.8 6.1 AD-69445 16.0 0.6 44.4 4.4 AD-69400 12.2 1.4 34.8 2.3 AD-69446 14.9 1.3 30.4 2.9 AD-69401 15.4 1.6 29.4 4.9 AD-69451 6.9 1.3 19.2 3.6 AD-69407 4.1 0.4 11.8 2.8 AD-1955 101.283 16.1184

TABLE 5 GCK Single Dose Screen in Primary Cynomologous Hepatocytes Primary Cyno Hepatocytes duplexName 10 nM Avg 10 nM SD 0.1 nM Avg 0.1 nM SD AD-69366 29.8 14.3 37.4 9.3 AD-69368 12.2 4.6 28.1 4.9 AD-69411 14.2 4.5 22.6 10.8 AD-69413 12.1 3.2 30.0 14.0 AD-69367 14.9 2.6 39.9 7.1 AD-69369 36.6 14.2 54.2 9.5 AD-69412 19.3 6.5 31.2 4.6 AD-69414 26.4 11.9 63.6 8.7 AD-69371 50.2 20.5 91.3 17.3 AD-69416 24.5 8.3 21.8 3.6 AD-69370 43.4 10.3 69.4 11.6 AD-69415 15.4 6.2 34.2 13.2 AD-69372 19.8 7.9 23.7 8.7 AD-69417 23.8 10.5 31.4 16.1 AD-69373 87.7 22.1 66.8 10.6 AD-69418 20.4 4.9 25.7 6.4 AD-69374 24.7 15.0 41.8 6.2 AD-69419 21.9 6.7 32.8 12.9 AD-69375 20.6 5.5 49.5 16.0 AD-69420 15.3 2.9 41.8 21.6 AD-69376 17.7 5.3 22.4 12.1 AD-69377 21.6 5.6 26.4 6.2 AD-69421 22.1 4.7 22.1 8.9 AD-69422 22.7 5.7 38.2 11.8 AD-69378 14.7 6.6 31.9 26.2 AD-69379 10.5 2.7 14.4 2.9 AD-69423 20.3 8.2 42.2 10.8 AD-69424 20.0 5.1 17.5 10.3 AD-69381 46.6 12.8 56.9 20.6 AD-69426 49.3 8.4 76.7 29.0 AD-69382 74.0 22.7 54.6 14.2 AD-69427 33.9 5.9 66.3 13.8 AD-69383 63.4 18.6 81.4 22.7 AD-69428 39.4 16.0 69.7 10.0 AD-69384 58.1 17.4 89.1 22.8 AD-69429 30.9 7.3 47.9 17.9 AD-69385 27.3 9.2 37.0 7.7 AD-69386 41.4 16.6 61.3 18.6 AD-69430 24.2 8.8 29.8 13.7 AD-69387 28.2 3.0 30.9 11.1 AD-69432 23.5 7.8 24.4 5.3 AD-69388 63.7 11.0 70.8 7.3 AD-69389 59.7 13.8 77.8 15.6 AD-69433 37.0 3.3 62.2 31.0 AD-69434 76.3 27.9 94.1 8.0 AD-69391 22.0 11.2 25.2 10.0 AD-69436 24.5 4.8 41.3 2.7 AD-69392 23.3 10.1 52.9 11.4 AD-69402 21.9 7.6 59.1 29.2 AD-69393 22.9 2.3 37.3 2.9 AD-69437 21.8 6.6 30.4 7.7 AD-69438 21.5 5.9 32.4 9.6 AD-69395 17.4 5.7 32.3 4.6 AD-69440 25.7 15.0 35.1 13.5 AD-69396 28.8 9.3 16.6 5.1 AD-69441 26.3 8.4 39.2 8.9 AD-69397 62.4 33.3 48.6 26.1 AD-69442 41.0 12.3 50.4 12.5 AD-69394 38.0 12.9 65.7 13.1 AD-69439 30.2 9.0 51.6 10.8 AD-69398 21.1 3.8 23.1 6.5 AD-69443 20.0 7.6 29.3 4.2 AD-69380 43.8 15.3 64.7 26.0 AD-69425 35.5 9.8 38.5 8.5 AD-69403 34.5 12.0 35.1 7.0 AD-69447 23.5 7.4 55.0 21.7 AD-69404 41.2 23.9 30.0 5.5 AD-69405 20.2 7.7 35.0 14.4 AD-69448 53.1 16.4 42.3 6.3 AD-69449 37.8 14.7 42.6 4.0 AD-69406 21.6 13.7 23.4 7.1 AD-69450 19.6 4.5 23.9 5.1 AD-69390 18.8 8.0 21.0 4.2 AD-69435 20.0 17.3 23.9 9.7 AD-69408 40.0 17.9 34.1 18.5 AD-69452 28.9 9.6 45.5 13.6 AD-69409 40.0 10.8 52.7 31.5 AD-69453 39.2 6.5 57.6 20.9 AD-69410 62.0 8.7 78.6 10.5 AD-69454 86.5 6.9 102.4 27.8 AD-69399 88.8 17.5 81.6 21.4 AD-69445 68.9 19.5 118.3 26.5 AD-69400 32.7 11.9 31.1 10.1 AD-69446 23.5 10.8 38.3 4.1 AD-69401 28.4 18.3 24.7 5.7 AD-69451 79.6 23.5 102.9 41.6 AD-69407 16.8 7.7 29.2 12.6 AD-1955 102.912 29.2078

Example 2. iRNA Design, Synthesis, Selection, and In Vitro Evaluation

This Example describes methods for the design, synthesis, selection, and in vitro evaluation of additional GCK iRNA agents.

Bioinformatics

A set of siRNAs targeting human glucokinase (GCK) (human NCBI refseqID: NM_033507; NCBI GeneID: 2645) were designed using custom R and Python scripts. The human GCK REFSEQ mRNA has a length of 2442 bases. The rationale and method for the set of siRNA designs is as follows: the predicted efficacy for every potential 19mer siRNA from position 10 through position 2442 was determined with a linear model derived the direct measure of mRNA knockdown from more than 20,000 distinct siRNA designs targeting a large number of vertebrate genes. The custom Python script built the set of siRNAs by systematically selecting an siRNA every 11 bases along the target mRNA nucleotide sequence starting at position 10. At each of the positions, the neighboring siRNA (one position to the 5′ end of the mRNA, one position to the 3′ end of the mRNA) was swapped into the design set if the predicted efficacy was better than the efficacy at the exact every-eleventh siRNA. Low complexity siRNAs, e.g., those with Shannon Entropy measures below 1.35, were excluded from the set.

Cell Culture and Transfections

Primary Cyno Hepatocytes (PCH) cells were transfected by adding 4.9 W of Opti-MEM plus 0.1 μl of Lipofectamine RNAiMax per well (Invitrogen, Carlsbad Calif. cat #13778-150) to 5 μl of siRNA duplexes per well into a 384-well plate and incubated at room temperature for 15 minutes. Forty μl of EMEM containing about 5×10³ cells were then added to the siRNA mixture. Cells were incubated for 24 hours prior to RNA purification. Single dose experiments were performed at 20 nM final duplex concentration.

Total RNA Isolation Using DYNABEADS mRNA Isolation Kit

RNA was isolated using an automated protocol on a BioTek-EL406 platform using DYNABEADs (Invitrogen, cat #61012). Briefly, 50 μl of Lysis/Binding Buffer and 25 μl of lysis buffer containing 3 μl of magnetic beads were added to the plate with cells. Plates were incubated on an electromagnetic shaker for 10 minutes at room temperature and then magnetic beads were captured and the supernatant was removed. Bead-bound RNA was then washed two times with 150 μl Wash Buffer A and once with Wash Buffer B. Beads were then washed with 150 μl Elution Buffer, re-captured, and the supernatant was removed.

cDNA Synthesis Using ABI High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, Calif., Cat #4368813)

Ten μl of a master mix containing 1 μl 10× Buffer, 0.4 μl 25× dNTPs, 1 μl 10× Random primers, 0.5 μl Reverse Transcriptase, 0.5 μl RNase inhibitor and 6.6 μl of H₂O per reaction was added to RNA isolated as described above. Plates were sealed, mixed, and incubated on an electromagnetic shaker for 10 minutes at room temperature, followed by 2 hours at 37° C.

Real Time PCR

Two μl of cDNA were added to a master mix containing 0.5 μl of Custom Cyno GAPDH TaqMan Probe, 0.5 μl cyno GCK probe (Mf02827184 ml) and 5 μl Lightcycler 480 probe master mix (Roche Cat #04887301001) per well in a 384 well plates (Roche cat #04887301001). Real time PCR was performed in a LightCycler480 Real Time PCR system (Roche) using the ΔΔCt(RQ) assay. Each duplex was tested in four independent transfections.

To calculate relative fold change, real time data were analyzed using the ΔΔCt method and normalized to assays performed with cells transfected with 20 nM AD-1955, or mock transfected cells.

A detailed list of the unmodified GCK sense and antisense strand sequences is shown in Table 6 and a detailed list of the modified GCK sense and antisense strand sequences is shown in Table 7.

Table 8 shows the results of a single dose screen in primary Cynomolgus hepatocytes (PCH) transfected with the indicated modified siRNAs. Data are expressed as percent of message remaining relative to cells treated with a non-targeting control siRNA, AD-1955.

TABLE 6 GCK Unmodified Sequences SEQ SEQ Duplex Sense Oligo ID Antissense ID Position in Name Name Sense Sequence (5′ to 3′) NO: Oligo Name Sense Sequence (5′ to 3′) NO: NM_033507.1 AD-71009 A-142377 CUGCCAGCCUCAGGCAGCU 476 A-142378 AGCUGCCUGAGGCUGGCAG 684 24-42 AD-71010 A-142379 UCAGGCAGCUCUCCAUCCA 477 A-142380 UGGAUGGAGAGCUGCCUGA 685 33-51 AD-71011 A-142381 CCAUCCAAGCAGCCGUUGA 478 A-142382 UCAACGGCUGCUUGGAUGG 686 45-63 AD-71012 A-142383 AGCCGUUGCUGCCACAGGA 479 A-142384 UCCUGUGGCAGCAACGGCU 687 55-73 AD-71013 A-142385 ACAGGCGGGCCUUACGCUA 480 A-142386 UAGCGUAAGGCCCGCCUGU 688 68-86 AD-71014 A-142387 UUACGCUCCAAGGCUACAA 481 A-142388 UUGUAGCCUUGGAGCGUAA 689 79-97 AD-71015 A-142389 AAGGCUACAGCAUGUGCUA 482 A-142390 UAGCACAUGCUGUAGCCUU 690  88-106 AD-71016 A-142391 UGUGCUAGGCCUCAGCAGA 483 A-142392 UCUGCUGAGGCCUAGCACA 691 100-118 AD-71017 A-142393 UCAGCAGGCAGGAGCAUCU 484 A-142394 AGAUGCUCCUGCCUGCUGA 692 111-129 AD-71018 A-142395 AGCAUCUCUGCCUCCCAAA 485 A-142396 UUUGGGAGGCAGAGAUGCU 693 123-141 AD-71019 A-142397 CCUCCCAAAGCAUCUACCU 486 A-142398 AGGUAGAUGCUUUGGGAGG 694 133-151 AD-71020 A-142401 UAGCCCCUCGGAGAGAUGA 487 A-142402 UCAUCUCUCCGAGGGGCUA 695 154-172 AD-71021 A-142403 AGAGAUGGCGAUGGAUGUA 488 A-142404 UACAUCCAUCGCCAUCUCU 696 165-183 AD-71022 A-142405 UGGAUGUCACAAGGAGCCA 489 A-142406 UGGCUCCUUGUGACAUCCA 697 176-194 AD-71023 A-142407 AGGAGCCAGGCCCAGACAA 490 A-142408 UUGUCUGGGCCUGGCUCCU 698 187-205 AD-71024 A-142411 ACUCUGGUAGAGCAGAUCA 491 A-142412 UGAUCUGCUCUACCAGAGU 699 211-229 AD-71025 A-142413 AGCAGAUCCUGGCAGAGUU 492 A-142414 AACUCUGCCAGGAUCUGCU 700 221-239 AD-71026 A-142415 CAGAGUUCCAGCUGCAGGA 493 A-142416 UCCUGCAGCUGGAACUCUG 701 233-251 AD-71027 A-142417 AGCUGCAGGAGGAGGACCU 494 A-142418 AGGUCCUCCUCCUGCAGCU 702 242-260 AD-71028 A-142419 AGGACCUGAAGAAGGUGAU 495 A-142420 AUCACCUUCUUCAGGUCCU 703 254-272 AD-71029 A-142421 AAGGUGAUGAGACGGAUGA 496 A-142422 UCAUCCGUCUCAUCACCUU 704 265-283 AD-71030 A-142423 CGGAUGCAGAAGGAGAUGA 497 A-142424 UCAUCUCCUUCUGCAUCCG 705 277-295 AD-71031 A-142425 AAGGAGAUGGACCGCGGCA 498 A-142426 UGCCGCGGUCCAUCUCCUU 706 286-304 AD-71032 A-142427 CGCGGCCUGAGGCUGGAGA 499 A-142428 UCUCCAGCCUCAGGCCGCG 707 298-316 AD-71033 A-142429 CUGGAGACCCAUGAAGAGA 500 A-142430 UCUCUUCAUGGGUCUCCAG 708 310-328 AD-71034 A-142431 CAUGAAGAGGCCAGUGUGA 501 A-142432 UCACACUGGCCUCUUCAUG 709 319-337 AD-71035 A-142433 CAGUGUGAAGAUGCUGCCA 502 A-142434 UGGCAGCAUCUUCACACUG 710 330-348 AD-71036 A-142435 UGCUGCCCACCUACGUGCA 503 A-142436 UGCACGUAGGUGGGCAGCA 711 341-359 AD-71037 A-142437 UACGUGCGCUCCACCCCAA 504 A-142438 UUGGGGUGGAGCGCACGUA 712 352-370 AD-71038 A-142439 ACCCCAGAAGGCUCAGAAA 505 A-142440 UUUCUGAGCCUUCUGGGGU 713 364-382 AD-71039 A-142441 UCAGAAGUCGGGGACUUCA 506 A-142442 UGAAGUCCCCGACUUCUGA 714 376-394 AD-71040 A-142443 GGGGACUUCCUCUCCCUGA 507 A-142444 UCAGGGAGAGGAAGUCCCC 715 385-403 AD-71041 A-142445 UCCCUGGACCUGGGUGGCA 508 A-142446 UGCCACCCAGGUCCAGGGA 716 397-415 AD-71042 A-142447 UGGGUGGCACUAACUUCAA 509 A-142448 UUGAAGUUAGUGCCACCCA 717 407-425 AD-71043 A-142449 ACUUCAGGGUGAUGCUGGU 510 A-142450 ACCAGCAUCACCCUGAAGU 718 419-437 AD-71044 A-142453 AGGUGGGAGAAGGUGAGGA 511 A-142454 UCCUCACCUUCUCCCACCU 719 440-458 AD-71045 A-142457 CAGUGGAGCGUGAAGACCA 512 A-142458 UGGUCUUCACGCUCCACUG 720 463-481 AD-71046 A-142461 CCAGAUGUACUCCAUCCCA 513 A-142462 UGGGAUGGAGUACAUCUGG 721 486-504 AD-71047 A-142467 ACCGGCACUGCUGAGAUGA 514 A-142468 UCAUCUCAGCAGUGCCGGU 722 517-535 AD-71048 A-142469 AGAUGCUCUUCGACUACAU 515 A-142470 AUGUAGUCGAAGAGCAUCU 723 530-548 AD-71049 A-142471 UCGACUACAUCUCUGAGUA 516 A-142472 UACUCAGAGAUGUAGUCGA 724 539-557 AD-71050 A-142473 UCUGAGUGCAUCUCCGACU 517 A-142474 AGUCGGAGAUGCACUCAGA 725 550-568 AD-71051 A-142475 UCCGACUUCCUGGACAAGA 518 A-142476 UCUUGUCCAGGAAGUCGGA 726 562-580 AD-71052 A-142477 GACAAGCAUCAGAUGAAAC 519 A-142478 GUUUCAUCUGAUGCUUGUC 727 574-592 AD-71053 A-142479 AGAUGAAACACAAGAAGCU 520 A-142480 AGCUUCUUGUGUUUCAUCU 728 584-602 AD-71054 A-142481 AGAAGCUGCCCCUGGGCUU 521 A-142482 AAGCCCAGGGGCAGCUUCU 729 596-614 AD-71055 A-142483 CCUGGGCUUCACCUUCUCA 522 A-142484 UGAGAAGGUGAAGCCCAGG 730 606-624 AD-71056 A-142485 ACCUUCUCCUUUCCUGUGA 523 A-142486 UCACAGGAAAGGAGAAGGU 731 616-634 AD-71057 A-142487 CUGUGAGGCACGAAGACAU 524 A-142488 AUGUCUUCGUGCCUCACAG 732 629-647 AD-71058 A-142489 GAAGACAUCGAUAAGGGCA 525 A-142490 UGCCCUUAUCGAUGUCUUC 733 640-658 AD-71059 A-142491 GAUAAGGGCAUCCUUCUCA 526 A-142492 UGAGAAGGAUGCCCUUAUC 734 649-667 AD-71060 A-142493 UUCUCAACUGGACCAAGGA 527 A-142494 UCCUUGGUCCAGUUGAGAA 735 662-680 AD-71061 A-142495 ACCAAGGGCUUCAAGGCCU 528 A-142496 AGGCCUUGAAGCCCUUGGU 736 673-691 AD-71062 A-142497 CAAGGCCUCAGGAGCAGAA 529 A-142498 UUCUGCUCCUGAGGCCUUG 737 684-702 AD-71063 A-142499 AGGAGCAGAAGGGAACAAU 530 A-142500 AUUGUUCCCUUCUGCUCCU 738 693-711 AD-71064 A-142501 AACAAUGUCGUGGGGCUUA 531 A-142502 UAAGCCCCACGACAUUGUU 739 706-724 AD-71065 A-142503 UGGGGCUUCUGCGAGACGA 532 A-142504 UCGUCUCGCAGAAGCCCCA 740 716-734 AD-71066 A-142505 CGAGACGCUAUCAAACGGA 533 A-142506 UCCGUUUGAUAGCGUCUCG 741 727-745 AD-71067 A-142507 AAACGGAGAGGGGACUUUA 534 A-142508 UAAAGUCCCCUCUCCGUUU 742 739-757 AD-71068 A-142509 GGGACUUUGAAAUGGAUGU 535 A-142510 ACAUCCAUUUCAAAGUCCC 743 749-767 AD-71069 A-142513 GCAAUGGUGAAUGACACGA 536 A-142514 UCGUGUCAUUCACCAUUGC 744 772-790 AD-71070 A-142515 AAUGACACGGUGGCCACGA 537 A-142516 UCGUGGCCACCGUGUCAUU 745 781-799 AD-71071 A-142517 GCCACGAUGAUCUCCUGCU 538 A-142518 AGCAGGAGAUCAUCGUGGC 746 793-811 AD-71072 A-142519 UCCUGCUACUACGAAGACA 539 A-142520 UGUCUUCGUAGUAGCAGGA 747 805-823 AD-71073 A-142523 AGUGCGAGGUCGGCAUGAU 540 A-142524 AUCAUGCCGACCUCGCACU 748 827-845 AD-71074 A-142525 GGCAUGAUCGUGGGCACGA 541 A-142526 UCGUGCCCACGAUCAUGCC 749 838-856 AD-71075 A-142527 GUGGGCACGGGCUGCAAUA 542 A-142528 UAUUGCAGCCCGUGCCCAC 750 847-865 AD-71076 A-142529 UGCAAUGCCUGCUACAUGA 543 A-142530 UCAUGUAGCAGGCAUUGCA 751 859-877 AD-71077 A-142531 UACAUGGAGGAGAUGCAGA 544 A-142532 UCUGCAUCUCCUCCAUGUA 752 871-889 AD-71078 A-142533 AGAUGCAGAAUGUGGAGCU 545 A-142534 AGCUCCACAUUCUGCAUCU 753 881-899 AD-71079 A-142535 UGUGGAGCUGGUGGAGGGA 546 A-142536 UCCCUCCACCAGCUCCACA 754 891-909 AD-71080 A-142537 UGGAGGGGGACGAGGGCCA 547 A-142538 UGGCCCUCGUCCCCCUCCA 755 902-920 AD-71081 A-142539 GAGGGCCGCAUGUGCGUCA 548 A-142540 UGACGCACAUGCGGCCCUC 756 913-931 AD-71082 A-142541 UGCGUCAAUACCGAGUGGA 549 A-142542 UCCACUCGGUAUUGACGCA 757 925-943 AD-71083 A-142543 CGAGUGGGGCGCCUUCGGA 550 A-142544 UCCGAAGGCGCCCCACUCG 758 936-954 AD-71084 A-142545 GCCUUCGGGGACUCCGGCA 551 A-142546 UGCCGGAGUCCCCGAAGGC 759 946-964 AD-71085 A-142547 UCCGGCGAGCUGGACGAGU 552 A-142548 ACUCGUCCAGCUCGCCGGA 760 958-976 AD-71086 A-142549 GACGAGUUCCUGCUGGAGU 553 A-142550 ACUCCAGCAGGAACUCGUC 761 970-988 AD-71087 A-142551 UGCUGGAGUAUGACCGCCU 554 A-142552 AGGCGGUCAUACUCCAGCA 762 980-998 AD-71088 A-142553 GACCGCCUGGUGGACGAGA 555 A-142554 UCUCGUCCACCAGGCGGUC 763  991-1009 AD-71089 A-142555 GGACGAGAGCUCUGCAAAC 556 A-142556 GUUUGCAGAGCUCUCGUCC 764 1002-1020 AD-71090 A-142557 UCUGCAAACCCCGGUCAGA 557 A-142558 UCUGACCGGGGUUUGCAGA 765 1012-1030 AD-71091 A-142559 GGUCAGCAGCUGUAUGAGA 558 A-142560 UCUCAUACAGCUGCUGACC 766 1024-1042 AD-71092 A-142561 UAUGAGAAGCUCAUAGGUA 559 A-142562 UACCUAUGAGCUUCUCAUA 767 1036-1054 AD-71093 A-142563 UCAUAGGUGGCAAGUACAU 560 A-142564 AUGUACUUGCCACCUAUGA 768 1046-1064 AD-71094 A-142565 AAGUACAUGGGCGAGCUGA 561 A-142566 UCAGCUCGCCCAUGUACUU 769 1057-1075 AD-71095 A-142567 GCGAGCUGGUGCGGCUUGU 562 A-142568 ACAAGCCGCACCAGCUCGC 770 1067-1085 AD-71096 A-142569 GGCUUGUGCUGCUCAGGCU 563 A-142570 AGCCUGAGCAGCACAAGCC 771 1079-1097 AD-71097 A-142571 UCAGGCUCGUGGACGAAAA 564 A-142572 UUUUCGUCCACGAGCCUGA 772 1091-1109 AD-69448 A-139832 UGGACGAAAACCUGCUCUU 565 A-139833 AAGAGCAGGUUUUCGUCCA 773 1100-1118 AD-71098 A-142573 UGCUCUUCCACGGGGAGGA 566 A-142574 UCCUCCCCGUGGAAGAGCA 774 1112-1130 AD-71099 A-142575 GGGAGGCCUCCGAGCAGCU 567 A-142576 AGCUGCUCGGAGGCCUCCC 775 1124-1142 AD-71100 A-142577 CGAGCAGCUGCGCACACGA 568 A-142578 UCGUGUGCGCAGCUGCUCG 776 1134-1152 AD-71101 A-142581 AGCCUUCGAGACGCGCUUA 569 A-142582 UAAGCGCGUCUCGAAGGCU 777 1155-1173 AD-71102 A-142583 CGCUUCGUGUCGCAGGUGA 570 A-142584 UCACCUGCGACACGAAGCG 778 1168-1186 AD-71103 A-142585 UCGCAGGUGGAGAGCGACA 571 A-142586 UGUCGCUCUCCACCUGCGA 779 1177-1195 AD-71104 A-142587 AGCGACACGGGCGACCGCA 572 A-142588 UGCGGUCGCCCGUGUCGCU 780 1189-1207 AD-71105 A-142589 CGACCGCAAGCAGAUCUAA 573 A-142590 UUAGAUCUGCUUGCGGUCG 781 1200-1218 AD-71106 A-142591 CAGAUCUACAACAUCCUGA 574 A-142592 UCAGGAUGUUGUAGAUCUG 782 1210-1228 AD-71107 A-142593 UCCUGAGCACGCUGGGGCU 575 A-142594 AGCCCCAGCGUGCUCAGGA 783 1223-1241 AD-71108 A-142595 CUGGGGCUGCGACCCUCGA 576 A-142596 UCGAGGGUCGCAGCCCCAG 784 1234-1252 AD-71109 A-142597 CGACCCUCGACCACCGACU 577 A-142598 AGUCGGUGGUCGAGGGUCG 785 1243-1261 AD-71110 A-142599 CACCGACUGCGACAUCGUA 578 A-142600 UACGAUGUCGCAGUCGGUG 786 1254-1272 AD-71111 A-142601 CAUCGUGCGCCGCGCCUGA 579 A-142602 UCAGGCGCGGCGCACGAUG 787 1266-1284 AD-71112 A-142603 CGCGCCUGCGAGAGCGUGU 580 A-142604 ACACGCUCUCGCAGGCGCG 788 1276-1294 AD-71113 A-142605 AGCGUGUCUACGCGCGCUA 581 A-142606 UAGCGCGCGUAGACACGCU 789 1288-1306 AD-71114 A-142607 CGCGCUGCGCACAUGUGCU 582 A-142608 AGCACAUGUGCGCAGCGCG 790 1300-1318 AD-71115 A-142609 ACAUGUGCUCGGCGGGGCU 583 A-142610 AGCCCCGCCGAGCACAUGU 791 1310-1328 AD-71116 A-142611 CGGGGCUGGCGGGCGUCAU 584 A-142612 AUGACGCCCGCCAGCCCCG 792 1322-1340 AD-71117 A-142613 CGGGCGUCAUCAACCGCAU 585 A-142614 AUGCGGUUGAUGACGCCCG 793 1331-1349 AD-71118 A-142615 AACCGCAUGCGCGAGAGCA 586 A-142616 UGCUCUCGCGCAUGCGGUU 794 1342-1360 AD-71119 A-142617 AGAGCCGCAGCGAGGACGU 587 A-142618 ACGUCCUCGCUGCGGCUCU 795 1355-1373 AD-71120 A-142619 CGAGGACGUAAUGCGCAUA 588 A-142620 UAUGCGCAUUACGUCCUCG 796 1365-1383 AD-71121 A-142621 UGCGCAUCACUGUGGGCGU 589 A-142622 ACGCCCACAGUGAUGCGCA 797 1376-1394 AD-71122 A-142623 UGGGCGUGGAUGGCUCCGU 590 A-142624 ACGGAGCCAUCCACGCCCA 798 1388-1406 AD-71123 A-142625 UGGCUCCGUGUACAAGCUA 591 A-142626 UAGCUUGUACACGGAGCCA 799 1398-1416 AD-71124 A-142627 UACAAGCUGCACCCCAGCU 592 A-142628 AGCUGGGGUGCAGCUUGUA 800 1408-1426 AD-71125 A-142629 CCAGCUUCAAGGAGCGGUU 593 A-142630 AACCGCUCCUUGAAGCUGG 801 1421-1439 AD-71126 A-142631 AGGAGCGGUUCCAUGCCAA 594 A-142632 UUGGCAUGGAACCGCUCCU 802 1430-1448 AD-71127 A-142633 AUGCCAGCGUGCGCAGGCU 595 A-142634 AGCCUGCGCACGCUGGCAU 803 1442-1460 AD-71128 A-142635 CGCAGGCUGACGCCCAGCU 596 A-142636 AGCUGGGCGUCAGCCUGCG 804 1453-1471 AD-71129 A-142637 CCCAGCUGCGAGAUCACCU 597 A-142638 AGGUGAUCUCGCAGCUGGG 805 1465-1483 AD-71130 A-142639 GAGAUCACCUUCAUCGAGU 598 A-142640 ACUCGAUGAAGGUGAUCUC 806 1474-1492 AD-71131 A-142641 AUCGAGUCGGAGGAGGGCA 599 A-142642 UGCCCUCCUCCGACUCGAU 807 1486-1504 AD-71132 A-142643 AGGAGGGCAGUGGCCGGGA 600 A-142644 UCCCGGCCACUGCCCUCCU 808 1496-1514 AD-71133 A-142645 CCGGGGCGCGGCCCUGGUA 601 A-142646 UACCAGGGCCGCGCCCCGG 809 1509-1527 AD-71134 A-142647 CCCUGGUCUCGGCGGUGGA 602 A-142648 UCCACCGCCGAGACCAGGG 810 1520-1538 AD-71135 A-142649 GCGGUGGCCUGUAAGAAGA 603 A-142650 UCUUCUUACAGGCCACCGC 811 1531-1549 AD-71136 A-142651 UAAGAAGGCCUGUAUGCUA 604 A-142652 UAGCAUACAGGCCUUCUUA 812 1542-1560 AD-71137 A-142653 CUGUAUGCUGGGCCAGUGA 605 A-142654 UCACUGGCCCAGCAUACAG 813 1551-1569 AD-71138 A-142655 CAGUGAGAGCAGUGGCCGA 606 A-142656 UCGGCCACUGCUCUCACUG 814 1564-1582 AD-71139 A-142657 CAGUGGCCGCAAGCGCAGA 607 A-142658 UCUGCGCUUGCGGCCACUG 815 1573-1591 AD-71140 A-142659 AGCGCAGGGAGGAUGCCAA 608 A-142660 UUGGCAUCCUCCCUGCGCU 816 1584-1602 AD-71141 A-142661 UGCCACAGCCCCACAGCAA 609 A-142662 UUGCUGUGGGGCUGUGGCA 817 1597-1615 AD-71142 A-142663 CACAGCACCCAGGCUCCAU 610 A-142664 AUGGAGCCUGGGUGCUGUG 818 1608-1626 AD-71143 A-142665 AGGCUCCAUGGGGAAGUGA 611 A-142666 UCACUUCCCCAUGGAGCCU 819 1618-1636 AD-71144 A-142667 GGAAGUGCUCCCCACACGU 612 A-142668 ACGUGUGGGGAGCACUUCC 820 1629-1647 AD-71145 A-142669 CCACACGUGCUCGCAGCCU 613 A-142670 AGGCUGCGAGCACGUGUGG 821 1640-1658 AD-71146 A-142671 UCGCAGCCUGGCGGGGCAA 614 A-142672 UUGCCCCGCCAGGCUGCGA 822 1650-1668 AD-71147 A-142673 CGGGGCAGGAGGCCUGGCA 615 A-142674 UGCCAGGCCUCCUGCCCCG 823 1661-1679 AD-71148 A-142675 CCUGGCCUUGUCAGGACCA 616 A-142676 UGGUCCUGACAAGGCCAGG 824 1673-1691 AD-71149 A-142677 CAGGACCCAGGCCGCCUGA 617 A-142678 UCAGGCGGCCUGGGUCCUG 825 1684-1702 AD-71150 A-142679 CCGCCUGCCAUACCGCUGA 618 A-142680 UCAGCGGUAUGGCAGGCGG 826 1695-1713 AD-71151 A-142681 UACCGCUGGGGAACAGAGA 619 A-142682 UCUCUGUUCCCCAGCGGUA 827 1705-1723 AD-71152 A-142683 AACAGAGCGGGCCUCUUCA 620 A-142684 UGAAGAGGCCCGCUCUGUU 828 1716-1734 AD-71153 A-142685 CUCUUCCCUCAGUUUUUCA 621 A-142686 UGAAAAACUGAGGGAAGAG 829 1728-1746 AD-71154 A-142687 UUUUUCGGUGGGACAGCCA 622 A-142688 UGGCUGUCCCACCGAAAAA 830 1740-1758 AD-71155 A-142689 GGGACAGCCCCAGGGCCCU 623 A-142690 AGGGCCCUGGGGCUGUCCC 831 1749-1767 AD-71156 A-142691 AGGGCCCUAACGGGGGUGA 624 A-142692 UCACCCCCGUUAGGGCCCU 832 1760-1778 AD-71157 A-142693 GGGUGCGGCAGGAGCAGGA 625 A-142694 UCCUGCUCCUGCCGCACCC 833 1773-1791 AD-71158 A-142695 AGGAGCAGGAACAGAGACU 626 A-142696 AGUCUCUGUUCCUGCUCCU 834 1782-1800 AD-71159 A-142697 AGAGACUCUGGAAGCCCCA 627 A-142698 UGGGGCUUCCAGAGUCUCU 835 1794-1812 AD-71160 A-142699 AAGCCCCCCACCUUUCUCA 628 A-142700 UGAGAAAGGUGGGGGGCUU 836 1805-1823 AD-71161 A-142701 UUUCUCGCUGGAAUCAAUU 629 A-142702 AAUUGAUUCCAGCGAGAAA 837 1817-1835 AD-71162 A-142703 AAUCAAUUUCCCAGAAGGA 630 A-142704 UCCUUCUGGGAAAUUGAUU 838 1828-1846 AD-71163 A-142705 CCCAGAAGGGAGUUGCUCA 631 A-142706 UGAGCAACUCCCUUCUGGG 839 1837-1855 AD-71164 A-142707 UUGCUCACUCAGGACUUUA 632 A-142708 UAAAGUCCUGAGUGAGCAA 840 1849-1867 AD-71165 A-142709 AGGACUUUGAUGCAUUUCA 633 A-142710 UGAAAUGCAUCAAAGUCCU 841 1859-1877 AD-71166 A-142711 AUUUCCACACUGUCAGAGA 634 A-142712 UCUCUGACAGUGUGGAAAU 842 1872-1890 AD-71167 A-142713 UGUCAGAGCUGUUGGCCUA 635 A-142714 UAGGCCAACAGCUCUGACA 843 1882-1900 AD-71168 A-142715 UUGGCCUCGCCUGGGCCCA 636 A-142716 UGGGCCCAGGCGAGGCCAA 844 1893-1911 AD-71169 A-142717 CUGGGCCCAGGCUCUGGGA 637 A-142718 UCCCAGAGCCUGGGCCCAG 845 1903-1921 AD-71170 A-142719 CUCUGGGAAGGGGUGCCCU 638 A-142720 AGGGCACCCCUUCCCAGAG 846 1914-1932 AD-71171 A-142721 UGCCCUCUGGAUCCUGCUA 639 A-142722 UAGCAGGAUCCAGAGGGCA 847 1927-1945 AD-71172 A-142723 UCCUGCUGUGGCCUCACUU 640 A-142724 AAGUGAGGCCACAGCAGGA 848 1938-1956 AD-71173 A-142725 CCUCACUUCCCUGGGAACU 641 A-142726 AGUUCCCAGGGAAGUGAGG 849 1949-1967 AD-71174 A-142727 CUGGGAACUCAUCCUGUGU 642 A-142728 ACACAGGAUGAGUUCCCAG 850 1959-1977 AD-71175 A-142729 CCUGUGUGGGGAGGCAGCU 643 A-142730 AGCUGCCUCCCCACACAGG 851 1971-1989 AD-71176 A-142731 GGAGGCAGCUCCAACAGCU 644 A-142732 AGCUGUUGGAGCUGCCUCC 852 1980-1998 AD-71177 A-142733 CAACAGCUUGACCAGACCU 645 A-142734 AGGUCUGGUCAAGCUGUUG 853 1991-2009 AD-71178 A-142735 CCAGACCUAGACCUGGGCA 646 A-142736 UGCCCAGGUCUAGGUCUGG 854 2002-2020 AD-71179 A-142737 CUGGGCCAAAAGGGCAGCA 647 A-142738 UGCUGCCCUUUUGGCCCAG 855 2014-2032 AD-71180 A-142739 AGGGCAGCCAGGGGCUGCU 648 A-142740 AGCAGCCCCUGGCUGCCCU 856 2024-2042 AD-71181 A-142741 GGGCUGCUCAUCACCCAGU 649 A-142742 ACUGGGUGAUGAGCAGCCC 857 2035-2053 AD-71182 A-142743 ACCCAGUCCUGGCCAUUUU 650 A-142744 AAAAUGGCCAGGACUGGGU 858 2047-2065 AD-71183 A-142745 GCCAUUUUCUUGCCUGAGA 651 A-142746 UCUCAGGCAAGAAAAUGGC 859 2058-2076 AD-71184 A-142747 CCUGAGGCUCAAGAGGCCA 652 A-142748 UGGCCUCUUGAGCCUCAGG 860 2070-2088 AD-71185 A-142749 AAGAGGCCCAGGGAGCAAU 653 A-142750 AUUGCUCCCUGGGCCUCUU 861 2080-2098 AD-71186 A-142751 GGAGCAAUGGGAGGGGGCU 654 A-142752 AGCCCCCUCCCAUUGCUCC 862 2091-2109 AD-71187 A-142753 AGGGGGCUCCAUGGAGGAA 655 A-142754 UUCCUCCAUGGAGCCCCCU 863 2102-2120 AD-71188 A-142755 GGAGGAGGUGUCCCAAGCU 656 A-142756 AGCUUGGGACACCUCCUCC 864 2114-2132 AD-71189 A-142757 UCCCAAGCUUUGAAUACCA 657 A-142758 UGGUAUUCAAAGCUUGGGA 865 2124-2142 AD-71190 A-142759 AAUACCCCCAGAGACCUUU 658 A-142760 AAAGGUCUCUGGGGGUAUU 866 2136-2154 AD-71191 A-142761 AGAGACCUUUUCUCUCCCA 659 A-142762 UGGGAGAGAAAAGGUCUCU 867 2145-2163 AD-71192 A-142763 UCUCCCAUACCAUCACUGA 660 A-142764 UCAGUGAUGGUAUGGGAGA 868 2157-2175 AD-71193 A-142765 UCACUGAGUGGCUUGUGAU 661 A-142766 AUCACAAGCCACUCAGUGA 869 2169-2187 AD-71194 A-142767 GGCUUGUGAUUCUGGGAUA 662 A-142768 UAUCCCAGAAUCACAAGCC 870 2178-2196 AD-71195 A-142769 UGGGAUGGACCCUCGCAGA 663 A-142770 UCUGCGAGGGUCCAUCCCA 871 2190-2208 AD-71196 A-142771 UCGCAGCAGGUGCAAGAGA 664 A-142772 UCUCUUGCACCUGCUGCGA 872 2202-2220 AD-71197 A-142773 UGCAAGAGACAGAGCCCCA 665 A-142774 UGGGGCUCUGUCUCUUGCA 873 2212-2230 AD-71198 A-142775 AGAGCCCCCAAGCCUCUGA 666 A-142776 UCAGAGGCUUGGGGGCUCU 874 2222-2240 AD-71199 A-142777 CUCUGCCCCAAGGGGCCCA 667 A-142778 UGGGCCCCUUGGGGCAGAG 875 2235-2253 AD-71200 A-142779 AAGGGGCCCACAAAGGGGA 668 A-142780 UCCCCUUUGUGGGCCCCUU 876 2244-2262 AD-71201 A-142781 AAAGGGGAGAAGGGCCAGA 669 A-142782 UCUGGCCCUUCUCCCCUUU 877 2255-2273 AD-71202 A-142783 GGGCCAGCCCUACAUCUUA 670 A-142784 UAAGAUGUAGGGCUGGCCC 878 2266-2284 AD-71203 A-142785 AUCUUCAGCUCCCAUAGCA 671 A-142786 UGCUAUGGGAGCUGAAGAU 879 2279-2297 AD-71204 A-142787 UCCCAUAGCGCUGGCUCAA 672 A-142788 UUGAGCCAGCGCUAUGGGA 880 2288-2306 AD-71205 A-142789 UGGCUCAGGAAGAAACCCA 673 A-142790 UGGGUUUCUUCCUGAGCCA 881 2299-2317 AD-71206 A-142791 AACCCCAAGCAGCAUUCAA 674 A-142792 UUGAAUGCUGCUUGGGGUU 882 2312-2330 AD-71207 A-142793 CAGCAUUCAGCACACCCCA 675 A-142794 UGGGGUGUGCUGAAUGCUG 883 2321-2339 AD-71208 A-142795 CACCCCAAGGGACAACCCA 676 A-142796 UGGGUUGUCCCUUGGGGUG 884 2333-2351 AD-71209 A-142797 ACAACCCCAUCAUAUGACA 677 A-142798 UGUCAUAUGAUGGGGUUGU 885 2344-2362 AD-71210 A-142801 ACCCUCUCCAUGCCCAACA 678 A-142802 UGUUGGGCAUGGAGAGGGU 886 2367-2385 AD-71211 A-142803 UGCCCAACCUAAGAUUGUA 679 A-142804 UACAAUCUUAGGUUGGGCA 887 2377-2395 AD-71212 A-142805 AAGAUUGUGUGGGUUUUUU 680 A-142806 AAAAAACCCACACAAUCUU 888 2387-2405 AD-71213 A-142807 UUUUUUAAUUAAAAAUGUU 681 A-142808 AACAUUUUUAAUUAAAAAA 889 2400-2418 AD-71214 A-142809 UAAAAAUGUUAAAAGUUUU 682 A-142810 AAAACUUUUAACAUUUUUA 890 2409-2427 AD-71215 A-142811 AAAGUUUUAAACAUGAAAA 683 A-142812 UUUUCAUGUUUAAAACUUU 891 2420-2438

TABLE 7 GCK Modified Sequences Duplex Sense Oligo Antissense Name Name Sense Sequence (5′-3′) SEQ ID NO: Oligo Name AD-71009 A-142377 CUGCCAGCCUCAGGCA 892 A-142378 GCUdTdT AD-71010 A-142379 UCAGGCAGCUCUCCAU 893 A-142380 CCAdTdT AD-71011 A-142381 CCAUCCAAGCAGCCGU 894 A-142382 UGAdTdT AD-71012 A-142383 AGCCGUUGCUGCCACA 895 A-142384 GGAdTdT AD-71013 A-142385 ACAGGCGGGCCUUACG 896 A-142386 CUAdTdT AD-71014 A-142387 UUACGCUCCAAGGCUA 897 A-142388 CAAdTdT AD-71015 A-142389 AAGGCUACAGCAUGUG 898 A-142390 CUAdTdT AD-71016 A-142391 UGUGCUAGGCCUCAGC 899 A-142392 AGAdTdT AD-71017 A-142393 UCAGCAGGCAGGAGCA 900 A-142394 UCUdTdT AD-71018 A-142395 AGCAUCUCUGCCUCCC 901 A-142396 AAAdTdT AD-71019 A-142397 CCUCCCAAAGCAUCUA 902 A-142398 CCUdTdT AD-71020 A-142401 UAGCCCCUCGGAGAGA 903 A-142402 UGAdTdT AD-71021 A-142403 AGAGAUGGCGAUGGAU 904 A-142404 GUAdTdT AD-71022 A-142405 UGGAUGUCACAAGGAG 905 A-142406 CCAdTdT AD-71023 A-142407 AGGAGCCAGGCCCAGA 906 A-142408 CAAdTdT AD-71024 A-142411 ACUCUGGUAGAGCAGA 907 A-142412 UCAdTdT AD-71025 A-142413 AGCAGAUCCUGGCAGA 908 A-142414 GUUdTdT AD-71026 A-142415 CAGAGUUCCAGCUGCA 909 A-142416 GGAdTdT AD-71027 A-142417 AGCUGCAGGAGGAGGA 910 A-142418 CCUdTdT AD-71028 A-142419 AGGACCUGAAGAAGGU 911 A-142420 GAUdTdT AD-71029 A-142421 AAGGUGAUGAGACGGA 912 A-142422 UGAdTdT AD-71030 A-142423 CGGAUGCAGAAGGAGA 913 A-142424 UGAdTdT AD-71031 A-142425 AAGGAGAUGGACCGCG 914 A-142426 GCAdTdT AD-71032 A-142427 CGCGGCCUGAGGCUGG 915 A-142428 AGAdTdT AD-71033 A-142429 CUGGAGACCCAUGAAG 916 A-142430 AGAdTdT AD-71034 A-142431 CAUGAAGAGGCCAGUG 917 A-142432 UGAdTdT AD-71035 A-142433 CAGUGUGAAGAUGCUG 918 A-142434 CCAdTdT AD-71036 A-142435 UGCUGCCCACCUACGU 919 A-142436 GCAdTdT AD-71037 A-142437 UACGUGCGCUCCACCCC 920 A-142438 AAdTdT AD-71038 A-142439 ACCCCAGAAGGCUCAG 921 A-142440 AAAdTdT AD-71039 A-142441 UCAGAAGUCGGGGACU 922 A-142442 UCAdTdT AD-71040 A-142443 GGGGACUUCCUCUCCC 923 A-142444 UGAdTdT AD-71041 A-142445 UCCCUGGACCUGGGUG 924 A-142446 GCAdTdT AD-71042 A-142447 UGGGUGGCACUAACUU 925 A-142448 CAAdTdT AD-71043 A-142449 ACUUCAGGGUGAUGCU 926 A-142450 GGUdTdT AD-71044 A-142453 AGGUGGGAGAAGGUGA 927 A-142454 GGAdTdT AD-71045 A-142457 CAGUGGAGCGUGAAGA 928 A-142458 CCAdTdT AD-71046 A-142461 CCAGAUGUACUCCAUC 929 A-142462 CCAdTdT AD-71047 A-142467 ACCGGCACUGCUGAGA 930 A-142468 UGAdTdT AD-71048 A-142469 AGAUGCUCUUCGACUA 931 A-142470 CAUdTdT AD-71049 A-142471 UCGACUACAUCUCUGA 932 A-142472 GUAdTdT AD-71050 A-142473 UCUGAGUGCAUCUCCG 933 A-142474 ACUdTdT AD-71051 A-142475 UCCGACUUCCUGGACA 934 A-142476 AGAdTdT AD-71052 A-142477 GACAAGCAUCAGAUGA 935 A-142478 AACdTdT AD-71053 A-142479 AGAUGAAACACAAGAA 936 A-142480 GCUdTdT AD-71054 A-142481 AGAAGCUGCCCCUGGG 937 A-142482 CUUdTdT AD-71055 A-142483 CCUGGGCUUCACCUUC 938 A-142484 UCAdTdT AD-71056 A-142485 ACCUUCUCCUUUCCUG 939 A-142486 UGAdTdT AD-71057 A-142487 CUGUGAGGCACGAAGA 940 A-142488 CAUdTdT AD-71058 A-142489 GAAGACAUCGAUAAGG 941 A-142490 GCAdTdT AD-71059 A-142491 GAUAAGGGCAUCCUUC 942 A-142492 UCAdTdT AD-71060 A-142493 UUCUCAACUGGACCAA 943 A-142494 GGAdTdT AD-71061 A-142495 ACCAAGGGCUUCAAGG 944 A-142496 CCUdTdT AD-71062 A-142497 CAAGGCCUCAGGAGCA 945 A-142498 GAAdTdT AD-71063 A-142499 AGGAGCAGAAGGGAAC 946 A-142500 AAUdTdT AD-71064 A-142501 AACAAUGUCGUGGGGC 947 A-142502 UUAdTdT AD-71065 A-142503 UGGGGCUUCUGCGAGA 948 A-142504 CGAdTdT AD-71066 A-142505 CGAGACGCUAUCAAAC 949 A-142506 GGAdTdT AD-71067 A-142507 AAACGGAGAGGGGACU 950 A-142508 UUAdTdT AD-71068 A-142509 GGGACUUUGAAAUGGA 951 A-142510 UGUdTdT AD-71069 A-142513 GCAAUGGUGAAUGACA 952 A-142514 CGAdTdT AD-71070 A-142515 AAUGACACGGUGGCCA 953 A-142516 CGAdTdT AD-71071 A-142517 GCCACGAUGAUCUCCU 954 A-142518 GCUdTdT AD-71072 A-142519 UCCUGCUACUACGAAG 955 A-142520 ACAdTdT AD-71073 A-142523 AGUGCGAGGUCGGCAU 956 A-142524 GAUdTdT AD-71074 A-142525 GGCAUGAUCGUGGGCA 957 A-142526 CGAdTdT AD-71075 A-142527 GUGGGCACGGGCUGCA 958 A-142528 AUAdTdT AD-71076 A-142529 UGCAAUGCCUGCUACA 959 A-142530 UGAdTdT AD-71077 A-142531 UACAUGGAGGAGAUGC 960 A-142532 AGAdTdT AD-71078 A-142533 AGAUGCAGAAUGUGGA 961 A-142534 GCUdTdT AD-71079 A-142535 UGUGGAGCUGGUGGAG 962 A-142536 GGAdTdT AD-71080 A-142537 UGGAGGGGGACGAGGG 963 A-142538 CCAdTdT AD-71081 A-142539 GAGGGCCGCAUGUGCG 964 A-142540 UCAdTdT AD-71082 A-142541 UGCGUCAAUACCGAGU 965 A-142542 GGAdTdT AD-71083 A-142543 CGAGUGGGGCGCCUUC 966 A-142544 GGAdTdT AD-71084 A-142545 GCCUUCGGGGACUCCG 967 A-142546 GCAdTdT AD-71085 A-142547 UCCGGCGAGCUGGACG 968 A-142548 AGUdTdT AD-71086 A-142549 GACGAGUUCCUGCUGG 969 A-142550 AGUdTdT AD-71087 A-142551 UGCUGGAGUAUGACCG 970 A-142552 CCUdTdT AD-71088 A-142553 GACCGCCUGGUGGACG 971 A-142554 AGAdTdT AD-71089 A-142555 GGACGAGAGCUCUGCA 972 A-142556 AACdTdT AD-71090 A-142557 UCUGCAAACCCCGGUC 973 A-142558 AGAdTdT AD-71091 A-142559 GGUCAGCAGCUGUAUG 974 A-142560 AGAdTdT AD-71092 A-142561 UAUGAGAAGCUCAUAG 975 A-142562 GUAdTdT AD-71093 A-142563 UCAUAGGUGGCAAGUA 976 A-142564 CAUdTdT AD-71094 A-142565 AAGUACAUGGGCGAGC 977 A-142566 UGAdTdT AD-71095 A-142567 GCGAGCUGGUGCGGCU 978 A-142568 UGUdTdT AD-71096 A-142569 GGCUUGUGCUGCUCAG 979 A-142570 GCUdTdT AD-71097 A-142571 UCAGGCUCGUGGACGA 980 A-142572 AAAdTdT AD-69448 A-139832 UGGACGAAAACCUGCU 981 A-139833 CUUdTdT AD-71098 A-142573 UGCUCUUCCACGGGGA 982 A-142574 GGAdTdT AD-71099 A-142575 GGGAGGCCUCCGAGCA 983 A-142576 GCUdTdT AD-71100 A-142577 CGAGCAGCUGCGCACA 984 A-142578 CGAdTdT AD-71101 A-142581 AGCCUUCGAGACGCGC 985 A-142582 UUAdTdT AD-71102 A-142583 CGCUUCGUGUCGCAGG 986 A-142584 UGAdTdT AD-71103 A-142585 UCGCAGGUGGAGAGCG 987 A-142586 ACAdTdT AD-71104 A-142587 AGCGACACGGGCGACC 988 A-142588 GCAdTdT AD-71105 A-142589 CGACCGCAAGCAGAUC 989 A-142590 UAAdTdT AD-71106 A-142591 CAGAUCUACAACAUCC 990 A-142592 UGAdTdT AD-71107 A-142593 UCCUGAGCACGCUGGG 991 A-142594 GCUdTdT AD-71108 A-142595 CUGGGGCUGCGACCCU 992 A-142596 CGAdTdT AD-71109 A-142597 CGACCCUCGACCACCGA 993 A-142598 CUdTdT AD-71110 A-142599 CACCGACUGCGACAUC 994 A-142600 GUAdTdT AD-71111 A-142601 CAUCGUGCGCCGCGCC 995 A-142602 UGAdTdT AD-71112 A-142603 CGCGCCUGCGAGAGCG 996 A-142604 UGUdTdT AD-71113 A-142605 AGCGUGUCUACGCGCG 997 A-142606 CUAdTdT AD-71114 A-142607 CGCGCUGCGCACAUGU 998 A-142608 GCUdTdT AD-71115 A-142609 ACAUGUGCUCGGCGGG 999 A-142610 GCUdTdT AD-71116 A-142611 CGGGGCUGGCGGGCGU 1000 A-142612 CAUdTdT AD-71117 A-142613 CGGGCGUCAUCAACCG 1001 A-142614 CAUdTdT AD-71118 A-142615 AACCGCAUGCGCGAGA 1002 A-142616 GCAdTdT AD-71119 A-142617 AGAGCCGCAGCGAGGA 1003 A-142618 CGUdTdT AD-71120 A-142619 CGAGGACGUAAUGCGC 1004 A-142620 AUAdTdT AD-71121 A-142621 UGCGCAUCACUGUGGG 1005 A-142622 CGUdTdT AD-71122 A-142623 UGGGCGUGGAUGGCUC 1006 A-142624 CGUdTdT AD-71123 A-142625 UGGCUCCGUGUACAAG 1007 A-142626 CUAdTdT AD-71124 A-142627 UACAAGCUGCACCCCA 1008 A-142628 GCUdTdT AD-71125 A-142629 CCAGCUUCAAGGAGCG 1009 A-142630 GUUdTdT AD-71126 A-142631 AGGAGCGGUUCCAUGC 1010 A-142632 CAAdTdT AD-71127 A-142633 AUGCCAGCGUGCGCAG 1011 A-142634 GCUdTdT AD-71128 A-142635 CGCAGGCUGACGCCCA 1012 A-142636 GCUdTdT AD-71129 A-142637 CCCAGCUGCGAGAUCA 1013 A-142638 CCUdTdT AD-71130 A-142639 GAGAUCACCUUCAUCG 1014 A-142640 AGUdTdT AD-71131 A-142641 AUCGAGUCGGAGGAGG 1015 A-142642 GCAdTdT AD-71132 A-142643 AGGAGGGCAGUGGCCG 1016 A-142644 GGAdTdT AD-71133 A-142645 CCGGGGCGCGGCCCUG 1017 A-142646 GUAdTdT AD-71134 A-142647 CCCUGGUCUCGGCGGU 1018 A-142648 GGAdTdT AD-71135 A-142649 GCGGUGGCCUGUAAGA 1019 A-142650 AGAdTdT AD-71136 A-142651 UAAGAAGGCCUGUAUG 1020 A-142652 CUAdTdT AD-71137 A-142653 CUGUAUGCUGGGCCAG 1021 A-142654 UGAdTdT AD-71138 A-142655 CAGUGAGAGCAGUGGC 1022 A-142656 CGAdTdT AD-71139 A-142657 CAGUGGCCGCAAGCGC 1023 A-142658 AGAdTdT AD-71140 A-142659 AGCGCAGGGAGGAUGC 1024 A-142660 CAAdTdT AD-71141 A-142661 UGCCACAGCCCCACAGC 1025 A-142662 AAdTdT AD-71142 A-142663 CACAGCACCCAGGCUCC 1026 A-142664 AUdTdT AD-71143 A-142665 AGGCUCCAUGGGGAAG 1027 A-142666 UGAdTdT AD-71144 A-142667 GGAAGUGCUCCCCACA 1028 A-142668 CGUdTdT AD-71145 A-142669 CCACACGUGCUCGCAG 1029 A-142670 CCUdTdT AD-71146 A-142671 UCGCAGCCUGGCGGGG 1030 A-142672 CAAdTdT AD-71147 A-142673 CGGGGCAGGAGGCCUG 1031 A-142674 GCAdTdT AD-71148 A-142675 CCUGGCCUUGUCAGGA 1032 A-142676 CCAdTdT AD-71149 A-142677 CAGGACCCAGGCCGCC 1033 A-142678 UGAdTdT AD-71150 A-142679 CCGCCUGCCAUACCGCU 1034 A-142680 GAdTdT AD-71151 A-142681 UACCGCUGGGGAACAG 1035 A-142682 AGAdTdT AD-71152 A-142683 AACAGAGCGGGCCUCU 1036 A-142684 UCAdTdT AD-71153 A-142685 CUCUUCCCUCAGUUUU 1037 A-142686 UCAdTdT AD-71154 A-142687 UUUUUCGGUGGGACAG 1038 A-142688 CCAdTdT AD-71155 A-142689 GGGACAGCCCCAGGGC 1039 A-142690 CCUdTdT AD-71156 A-142691 AGGGCCCUAACGGGGG 1040 A-142692 UGAdTdT AD-71157 A-142693 GGGUGCGGCAGGAGCA 1041 A-142694 GGAdTdT AD-71158 A-142695 AGGAGCAGGAACAGAG 1042 A-142696 ACUdTdT AD-71159 A-142697 AGAGACUCUGGAAGCC 1043 A-142698 CCAdTdT AD-71160 A-142699 AAGCCCCCCACCUUUCU 1044 A-142700 CAdTdT AD-71161 A-142701 UUUCUCGCUGGAAUCA 1045 A-142702 AUUdTdT AD-71162 A-142703 AAUCAAUUUCCCAGAA 1046 A-142704 GGAdTdT AD-71163 A-142705 CCCAGAAGGGAGUUGC 1047 A-142706 UCAdTdT AD-71164 A-142707 UUGCUCACUCAGGACU 1048 A-142708 UUAdTdT AD-71165 A-142709 AGGACUUUGAUGCAUU 1049 A-142710 UCAdTdT AD-71166 A-142711 AUUUCCACACUGUCAG 1050 A-142712 AGAdTdT AD-71167 A-142713 UGUCAGAGCUGUUGGC 1051 A-142714 CUAdTdT AD-71168 A-142715 UUGGCCUCGCCUGGGC 1052 A-142716 CCAdTdT AD-71169 A-142717 CUGGGCCCAGGCUCUG 1053 A-142718 GGAdTdT AD-71170 A-142719 CUCUGGGAAGGGGUGC 1054 A-142720 CCUdTdT AD-71171 A-142721 UGCCCUCUGGAUCCUG 1055 A-142722 CUAdTdT AD-71172 A-142723 UCCUGCUGUGGCCUCA 1056 A-142724 CUUdTdT AD-71173 A-142725 CCUCACUUCCCUGGGA 1057 A-142726 ACUdTdT AD-71174 A-142727 CUGGGAACUCAUCCUG 1058 A-142728 UGUdTdT AD-71175 A-142729 CCUGUGUGGGGAGGCA 1059 A-142730 GCUdTdT AD-71176 A-142731 GGAGGCAGCUCCAACA 1060 A-142732 GCUdTdT AD-71177 A-142733 CAACAGCUUGACCAGA 1061 A-142734 CCUdTdT AD-71178 A-142735 CCAGACCUAGACCUGG 1062 A-142736 GCAdTdT AD-71179 A-142737 CUGGGCCAAAAGGGCA 1063 A-142738 GCAdTdT AD-71180 A-142739 AGGGCAGCCAGGGGCU 1064 A-142740 GCUdTdT AD-71181 A-142741 GGGCUGCUCAUCACCC 1065 A-142742 AGUdTdT AD-71182 A-142743 ACCCAGUCCUGGCCAU 1066 A-142744 UUUdTdT AD-71183 A-142745 GCCAUUUUCUUGCCUG 1067 A-142746 AGAdTdT AD-71184 A-142747 CCUGAGGCUCAAGAGG 1068 A-142748 CCAdTdT AD-71185 A-142749 AAGAGGCCCAGGGAGC 1069 A-142750 AAUdTdT AD-71186 A-142751 GGAGCAAUGGGAGGGG 1070 A-142752 GCUdTdT AD-71187 A-142753 AGGGGGCUCCAUGGAG 1071 A-142754 GAAdTdT AD-71188 A-142755 GGAGGAGGUGUCCCAA 1072 A-142756 GCUdTdT AD-71189 A-142757 UCCCAAGCUUUGAAUA 1073 A-142758 CCAdTdT AD-71190 A-142759 AAUACCCCCAGAGACC 1074 A-142760 UUUdTdT AD-71191 A-142761 AGAGACCUUUUCUCUC 1075 A-142762 CCAdTdT AD-71192 A-142763 UCUCCCAUACCAUCAC 1076 A-142764 UGAdTdT AD-71193 A-142765 UCACUGAGUGGCUUGU 1077 A-142766 GAUdTdT AD-71194 A-142767 GGCUUGUGAUUCUGGG 1078 A-142768 AUAdTdT AD-71195 A-142769 UGGGAUGGACCCUCGC 1079 A-142770 AGAdTdT AD-71196 A-142771 UCGCAGCAGGUGCAAG 1080 A-142772 AGAdTdT AD-71197 A-142773 UGCAAGAGACAGAGCC 1081 A-142774 CCAdTdT AD-71198 A-142775 AGAGCCCCCAAGCCUC 1082 A-142776 UGAdTdT AD-71199 A-142777 CUCUGCCCCAAGGGGC 1083 A-142778 CCAdTdT AD-71200 A-142779 AAGGGGCCCACAAAGG 1084 A-142780 GGAdTdT AD-71201 A-142781 AAAGGGGAGAAGGGCC 1085 A-142782 AGAdTdT AD-71202 A-142783 GGGCCAGCCCUACAUC 1086 A-142784 UUAdTdT AD-71203 A-142785 AUCUUCAGCUCCCAUA 1087 A-142786 GCAdTdT AD-71204 A-142787 UCCCAUAGCGCUGGCU 1088 A-142788 CAAdTdT AD-71205 A-142789 UGGCUCAGGAAGAAAC 1089 A-142790 CCAdTdT AD-71206 A-142791 AACCCCAAGCAGCAUU 1090 A-142792 CAAdTdT AD-71207 A-142793 CAGCAUUCAGCACACC 1091 A-142794 CCAdTdT AD-71208 A-142795 CACCCCAAGGGACAAC 1092 A-142796 CCAdTdT AD-71209 A-142797 ACAACCCCAUCAUAUG 1093 A-142798 ACAdTdT AD-71210 A-142801 ACCCUCUCCAUGCCCAA 1094 A-142802 CAdTdT AD-71211 A-142803 UGCCCAACCUAAGAUU 1095 A-142804 GUAdTdT AD-71212 A-142805 AAGAUUGUGUGGGUUU 1096 A-142806 UUUdTdT AD-71213 A-142807 UUUUUUAAUUAAAAAU 1097 A-142808 GUUdTdT AD-71214 A-142809 UAAAAAUGUUAAAAGU 1098 A-142810 UUUdTdT AD-71215 A-142811 AAAGUUUUAAACAUGA 1099 A-142812 AAAdTdT SEQ SEQ Duplex ID ID Name Antisense Sequence (5′-3′) NO: mRNA target sequence NO: AD-71009 AGCUGCCUGAGGCUGG 1100 CUGCCAGCCUCAGGCA 1308 CAGdTdT GCU AD-71010 UGGAUGGAGAGCUGCC 1101 UCAGGCAGCUCUCCAU 1309 UGAdTdT CCA AD-71011 UCAACGGCUGCUUGGA 1102 CCAUCCAAGCAGCCGU 1310 UGGdTdT UGC AD-71012 UCCUGUGGCAGCAACG 1103 AGCCGUUGCUGCCACA 1311 GCUdTdT GGC AD-71013 UAGCGUAAGGCCCGCC 1104 ACAGGCGGGCCUUACG 1312 UGUdTdT CUC AD-71014 UUGUAGCCUUGGAGCG 1105 UUACGCUCCAAGGCUA 1313 UAAdTdT CAG AD-71015 UAGCACAUGCUGUAGC 1106 AAGGCUACAGCAUGUG 1314 CUUdTdT CUA AD-71016 UCUGCUGAGGCCUAGC 1107 UGUGCUAGGCCUCAGC 1315 ACAdTdT AGG AD-71017 AGAUGCUCCUGCCUGC 1108 UCAGCAGGCAGGAGCA 1316 UGAdTdT UCU AD-71018 UUUGGGAGGCAGAGAU 1109 AGCAUCUCUGCCUCCC 1317 GCUdTdT AAA AD-71019 AGGUAGAUGCUUUGGG 1110 CCUCCCAAAGCAUCUA 1318 AGGdTdT CCU AD-71020 UCAUCUCUCCGAGGGG 1111 UAGCCCCUCGGAGAGA 1319 CUAdTdT UGG AD-71021 UACAUCCAUCGCCAUC 1112 AGAGAUGGCGAUGGAU 1320 UCUdTdT GUC AD-71022 UGGCUCCUUGUGACAU 1113 UGGAUGUCACAAGGAG 1321 CCAdTdT CCA AD-71023 UUGUCUGGGCCUGGCU 1114 AGGAGCCAGGCCCAGA 1322 CCUdTdT CAG AD-71024 UGAUCUGCUCUACCAG 1115 ACUCUGGUAGAGCAGA 1323 AGUdTdT UCC AD-71025 AACUCUGCCAGGAUCU 1116 AGCAGAUCCUGGCAGA 1324 GCUdTdT GUU AD-71026 UCCUGCAGCUGGAACU 1117 CAGAGUUCCAGCUGCA 1325 CUGdTdT GGA AD-71027 AGGUCCUCCUCCUGCA 1118 AGCUGCAGGAGGAGGA 1326 GCUdTdT CCU AD-71028 AUCACCUUCUUCAGGU 1119 AGGACCUGAAGAAGGU 1327 CCUdTdT GAU AD-71029 UCAUCCGUCUCAUCAC 1120 AAGGUGAUGAGACGGA 1328 CUUdTdT UGC AD-71030 UCAUCUCCUUCUGCAU 1121 CGGAUGCAGAAGGAGA 1329 CCGdTdT UGG AD-71031 UGCCGCGGUCCAUCUC 1122 AAGGAGAUGGACCGCG 1330 CUUdTdT GCC AD-71032 UCUCCAGCCUCAGGCC 1123 CGCGGCCUGAGGCUGG 1331 GCGdTdT AGA AD-71033 UCUCUUCAUGGGUCUC 1124 CUGGAGACCCAUGAAG 1332 CAGdTdT AGG AD-71034 UCACACUGGCCUCUUC 1125 CAUGAAGAGGCCAGUG 1333 AUGdTdT UGA AD-71035 UGGCAGCAUCUUCACA 1126 CAGUGUGAAGAUGCUG 1334 CUGdTdT CCC AD-71036 UGCACGUAGGUGGGCA 1127 UGCUGCCCACCUACGU 1335 GCAdTdT GCG AD-71037 UUGGGGUGGAGCGCAC 1128 UACGUGCGCUCCACCCC 1336 GUAdTdT AG AD-71038 UUUCUGAGCCUUCUGG 1129 ACCCCAGAAGGCUCAG 1337 GGUdTdT AAG AD-71039 UGAAGUCCCCGACUUC 1130 UCAGAAGUCGGGGACU 1338 UGAdTdT UCC AD-71040 UCAGGGAGAGGAAGUC 1131 GGGGACUUCCUCUCCC 1339 CCCdTdT UGG AD-71041 UGCCACCCAGGUCCAG 1132 UCCCUGGACCUGGGUG 1340 GGAdTdT GCA AD-71042 UUGAAGUUAGUGCCAC 1133 UGGGUGGCACUAACUU 1341 CCAdTdT CAG AD-71043 ACCAGCAUCACCCUGA 1134 ACUUCAGGGUGAUGCU 1342 AGUdTdT GGU AD-71044 UCCUCACCUUCUCCCAC 1135 AGGUGGGAGAAGGUGA 1343 CUdTdT GGA AD-71045 UGGUCUUCACGCUCCA 1136 CAGUGGAGCGUGAAGA 1344 CUGdTdT CCA AD-71046 UGGGAUGGAGUACAUC 1137 CCAGAUGUACUCCAUC 1345 UGGdTdT CCC AD-71047 UCAUCUCAGCAGUGCC 1138 ACCGGCACUGCUGAGA 1346 GGUdTdT UGC AD-71048 AUGUAGUCGAAGAGCA 1139 AGAUGCUCUUCGACUA 1347 UCUdTdT CAU AD-71049 UACUCAGAGAUGUAGU 1140 UCGACUACAUCUCUGA 1348 CGAdTdT GUG AD-71050 AGUCGGAGAUGCACUC 1141 UCUGAGUGCAUCUCCG 1349 AGAdTdT ACU AD-71051 UCUUGUCCAGGAAGUC 1142 UCCGACUUCCUGGACA 1350 GGAdTdT AGC AD-71052 GUUUCAUCUGAUGCUU 1143 GACAAGCAUCAGAUGA 1351 GUCdTdT AAC AD-71053 AGCUUCUUGUGUUUCA 1144 AGAUGAAACACAAGAA 1352 UCUdTdT GCU AD-71054 AAGCCCAGGGGCAGCU 1145 AGAAGCUGCCCCUGGG 1353 UCUdTdT CUU AD-71055 UGAGAAGGUGAAGCCC 1146 CCUGGGCUUCACCUUC 1354 AGGdTdT UCC AD-71056 UCACAGGAAAGGAGAA 1147 ACCUUCUCCUUUCCUG 1355 GGUdTdT UGA AD-71057 AUGUCUUCGUGCCUCA 1148 CUGUGAGGCACGAAGA 1356 CAGdTdT CAU AD-71058 UGCCCUUAUCGAUGUC 1149 GAAGACAUCGAUAAGG 1357 UUCdTdT GCA AD-71059 UGAGAAGGAUGCCCUU 1150 GAUAAGGGCAUCCUUC 1358 AUCdTdT UCA AD-71060 UCCUUGGUCCAGUUGA 1151 UUCUCAACUGGACCAA 1359 GAAdTdT GGG AD-71061 AGGCCUUGAAGCCCUU 1152 ACCAAGGGCUUCAAGG 1360 GGUdTdT CCU AD-71062 UUCUGCUCCUGAGGCC 1153 CAAGGCCUCAGGAGCA 1361 UUGdTdT GAA AD-71063 AUUGUUCCCUUCUGCU 1154 AGGAGCAGAAGGGAAC 1362 CCUdTdT AAU AD-71064 UAAGCCCCACGACAUU 1155 AACAAUGUCGUGGGGC 1363 GUUdTdT UUC AD-71065 UCGUCUCGCAGAAGCC 1156 UGGGGCUUCUGCGAGA 1364 CCAdTdT CGC AD-71066 UCCGUUUGAUAGCGUC 1157 CGAGACGCUAUCAAAC 1365 UCGdTdT GGA AD-71067 UAAAGUCCCCUCUCCG 1158 AAACGGAGAGGGGACU 1366 UUUdTdT UUG AD-71068 ACAUCCAUUUCAAAGU 1159 GGGACUUUGAAAUGGA 1367 CCCdTdT UGU AD-71069 UCGUGUCAUUCACCAU 1160 GCAAUGGUGAAUGACA 1368 UGCdTdT CGG AD-71070 UCGUGGCCACCGUGUC 1161 AAUGACACGGUGGCCA 1369 AUUdTdT CGA AD-71071 AGCAGGAGAUCAUCGU 1162 GCCACGAUGAUCUCCU 1370 GGCdTdT GCU AD-71072 UGUCUUCGUAGUAGCA 1163 UCCUGCUACUACGAAG 1371 GGAdTdT ACC AD-71073 AUCAUGCCGACCUCGC 1164 AGUGCGAGGUCGGCAU 1372 ACUdTdT GAU AD-71074 UCGUGCCCACGAUCAU 1165 GGCAUGAUCGUGGGCA 1373 GCCdTdT CGG AD-71075 UAUUGCAGCCCGUGCC 1166 GUGGGCACGGGCUGCA 1374 CACdTdT AUG AD-71076 UCAUGUAGCAGGCAUU 1167 UGCAAUGCCUGCUACA 1375 GCAdTdT UGG AD-71077 UCUGCAUCUCCUCCAU 1168 UACAUGGAGGAGAUGC 1376 GUAdTdT AGA AD-71078 AGCUCCACAUUCUGCA 1169 AGAUGCAGAAUGUGGA 1377 UCUdTdT GCU AD-71079 UCCCUCCACCAGCUCCA 1170 UGUGGAGCUGGUGGAG 1378 CAdTdT GGG AD-71080 UGGCCCUCGUCCCCCUC 1171 UGGAGGGGGACGAGGG 1379 CAdTdT CCG AD-71081 UGACGCACAUGCGGCC 1172 GAGGGCCGCAUGUGCG 1380 CUCdTdT UCA AD-71082 UCCACUCGGUAUUGAC 1173 UGCGUCAAUACCGAGU 1381 GCAdTdT GGG AD-71083 UCCGAAGGCGCCCCAC 1174 CGAGUGGGGCGCCUUC 1382 UCGdTdT GGG AD-71084 UGCCGGAGUCCCCGAA 1175 GCCUUCGGGGACUCCG 1383 GGCdTdT GCG AD-71085 ACUCGUCCAGCUCGCC 1176 UCCGGCGAGCUGGACG 1384 GGAdTdT AGU AD-71086 ACUCCAGCAGGAACUC 1177 GACGAGUUCCUGCUGG 1385 GUCdTdT AGU AD-71087 AGGCGGUCAUACUCCA 1178 UGCUGGAGUAUGACCG 1386 GCAdTdT CCU AD-71088 UCUCGUCCACCAGGCG 1179 GACCGCCUGGUGGACG 1387 GUCdTdT AGA AD-71089 GUUUGCAGAGCUCUCG 1180 GGACGAGAGCUCUGCA 1388 UCCdTdT AAC AD-71090 UCUGACCGGGGUUUGC 1181 UCUGCAAACCCCGGUC 1389 AGAdTdT AGC AD-71091 UCUCAUACAGCUGCUG 1182 GGUCAGCAGCUGUAUG 1390 ACCdTdT AGA AD-71092 UACCUAUGAGCUUCUC 1183 UAUGAGAAGCUCAUAG 1391 AUAdTdT GUG AD-71093 AUGUACUUGCCACCUA 1184 UCAUAGGUGGCAAGUA 1392 UGAdTdT CAU AD-71094 UCAGCUCGCCCAUGUA 1185 AAGUACAUGGGCGAGC 1393 CUUdTdT UGG AD-71095 ACAAGCCGCACCAGCU 1186 GCGAGCUGGUGCGGCU 1394 CGCdTdT UGU AD-71096 AGCCUGAGCAGCACAA 1187 GGCUUGUGCUGCUCAG 1395 GCCdTdT GCU AD-71097 UUUUCGUCCACGAGCC 1188 UCAGGCUCGUGGACGA 1396 UGAdTdT AAA AD-69448 AAGAGCAGGUUUUCGU 1189 UGGACGAAAACCUGCU 1397 CCAdTdT CUU AD-71098 UCCUCCCCGUGGAAGA 1190 UGCUCUUCCACGGGGA 1398 GCAdTdT GGC AD-71099 AGCUGCUCGGAGGCCU 1191 GGGAGGCCUCCGAGCA 1399 CCCdTdT GCU AD-71100 UCGUGUGCGCAGCUGC 1192 CGAGCAGCUGCGCACA 1400 UCGdTdT CGC AD-71101 UAAGCGCGUCUCGAAG 1193 AGCCUUCGAGACGCGC 1401 GCUdTdT UUC AD-71102 UCACCUGCGACACGAA 1194 CGCUUCGUGUCGCAGG 1402 GCGdTdT UGG AD-71103 UGUCGCUCUCCACCUG 1195 UCGCAGGUGGAGAGCG 1403 CGAdTdT ACA AD-71104 UGCGGUCGCCCGUGUC 1196 AGCGACACGGGCGACC 1404 GCUdTdT GCA AD-71105 UUAGAUCUGCUUGCGG 1197 CGACCGCAAGCAGAUC 1405 UCGdTdT UAC AD-71106 UCAGGAUGUUGUAGAU 1198 CAGAUCUACAACAUCC 1406 CUGdTdT UGA AD-71107 AGCCCCAGCGUGCUCA 1199 UCCUGAGCACGCUGGG 1407 GGAdTdT GCU AD-71108 UCGAGGGUCGCAGCCC 1200 CUGGGGCUGCGACCCU 1408 CAGdTdT CGA AD-71109 AGUCGGUGGUCGAGGG 1201 CGACCCUCGACCACCGA 1409 UCGdTdT CU AD-71110 UACGAUGUCGCAGUCG 1202 CACCGACUGCGACAUC 1410 GUGdTdT GUG AD-71111 UCAGGCGCGGCGCACG 1203 CAUCGUGCGCCGCGCC 1411 AUGdTdT UGC AD-71112 ACACGCUCUCGCAGGC 1204 CGCGCCUGCGAGAGCG 1412 GCGdTdT UGU AD-71113 UAGCGCGCGUAGACAC 1205 AGCGUGUCUACGCGCG 1413 GCUdTdT CUG AD-71114 AGCACAUGUGCGCAGC 1206 CGCGCUGCGCACAUGU 1414 GCGdTdT GCU AD-71115 AGCCCCGCCGAGCACA 1207 ACAUGUGCUCGGCGGG 1415 UGUdTdT GCU AD-71116 AUGACGCCCGCCAGCCC 1208 CGGGGCUGGCGGGCGU 1416 CGdTdT CAU AD-71117 AUGCGGUUGAUGACGC 1209 CGGGCGUCAUCAACCG 1417 CCGdTdT CAU AD-71118 UGCUCUCGCGCAUGCG 1210 AACCGCAUGCGCGAGA 1418 GUUdTdT GCC AD-71119 ACGUCCUCGCUGCGGC 1211 AGAGCCGCAGCGAGGA 1419 UCUdTdT CGU AD-71120 UAUGCGCAUUACGUCC 1212 CGAGGACGUAAUGCGC 1420 UCGdTdT AUC AD-71121 ACGCCCACAGUGAUGC 1213 UGCGCAUCACUGUGGG 1421 GCAdTdT CGU AD-71122 ACGGAGCCAUCCACGC 1214 UGGGCGUGGAUGGCUC 1422 CCAdTdT CGU AD-71123 UAGCUUGUACACGGAG 1215 UGGCUCCGUGUACAAG 1423 CCAdTdT CUG AD-71124 AGCUGGGGUGCAGCUU 1216 UACAAGCUGCACCCCA 1424 GUAdTdT GCU AD-71125 AACCGCUCCUUGAAGC 1217 CCAGCUUCAAGGAGCG 1425 UGGdTdT GUU AD-71126 UUGGCAUGGAACCGCU 1218 AGGAGCGGUUCCAUGC 1426 CCUdTdT CAG AD-71127 AGCCUGCGCACGCUGG 1219 AUGCCAGCGUGCGCAG 1427 CAUdTdT GCU AD-71128 AGCUGGGCGUCAGCCU 1220 CGCAGGCUGACGCCCA 1428 GCGdTdT GCU AD-71129 AGGUGAUCUCGCAGCU 1221 CCCAGCUGCGAGAUCA 1429 GGGdTdT CCU AD-71130 ACUCGAUGAAGGUGAU 1222 GAGAUCACCUUCAUCG 1430 CUCdTdT AGU AD-71131 UGCCCUCCUCCGACUCG 1223 AUCGAGUCGGAGGAGG 1431 AUdTdT GCA AD-71132 UCCCGGCCACUGCCCUC 1224 AGGAGGGCAGUGGCCG 1432 CUdTdT GGG AD-71133 UACCAGGGCCGCGCCCC 1225 CCGGGGCGCGGCCCUG 1433 GGdTdT GUC AD-71134 UCCACCGCCGAGACCA 1226 CCCUGGUCUCGGCGGU 1434 GGGdTdT GGC AD-71135 UCUUCUUACAGGCCAC 1227 GCGGUGGCCUGUAAGA 1435 CGCdTdT AGG AD-71136 UAGCAUACAGGCCUUC 1228 UAAGAAGGCCUGUAUG 1436 UUAdTdT CUG AD-71137 UCACUGGCCCAGCAUA 1229 CUGUAUGCUGGGCCAG 1437 CAGdTdT UGA AD-71138 UCGGCCACUGCUCUCA 1230 CAGUGAGAGCAGUGGC 1438 CUGdTdT CGC AD-71139 UCUGCGCUUGCGGCCA 1231 CAGUGGCCGCAAGCGC 1439 CUGdTdT AGG AD-71140 UUGGCAUCCUCCCUGC 1232 AGCGCAGGGAGGAUGC 1440 GCUdTdT CAC AD-71141 UUGCUGUGGGGCUGUG 1233 UGCCACAGCCCCACAGC 1441 GCAdTdT AC AD-71142 AUGGAGCCUGGGUGCU 1234 CACAGCACCCAGGCUCC 1442 GUGdTdT AU AD-71143 UCACUUCCCCAUGGAG 1235 AGGCUCCAUGGGGAAG 1443 CCUdTdT UGC AD-71144 ACGUGUGGGGAGCACU 1236 GGAAGUGCUCCCCACA 1444 UCCdTdT CGU AD-71145 AGGCUGCGAGCACGUG 1237 CCACACGUGCUCGCAG 1445 UGGdTdT CCU AD-71146 UUGCCCCGCCAGGCUG 1238 UCGCAGCCUGGCGGGG 1446 CGAdTdT CAG AD-71147 UGCCAGGCCUCCUGCCC 1239 CGGGGCAGGAGGCCUG 1447 CGdTdT GCC AD-71148 UGGUCCUGACAAGGCC 1240 CCUGGCCUUGUCAGGA 1448 AGGdTdT CCC AD-71149 UCAGGCGGCCUGGGUC 1241 CAGGACCCAGGCCGCC 1449 CUGdTdT UGC AD-71150 UCAGCGGUAUGGCAGG 1242 CCGCCUGCCAUACCGCU 1450 CGGdTdT GG AD-71151 UCUCUGUUCCCCAGCG 1243 UACCGCUGGGGAACAG 1451 GUAdTdT AGC AD-71152 UGAAGAGGCCCGCUCU 1244 AACAGAGCGGGCCUCU 1452 GUUdTdT UCC AD-71153 UGAAAAACUGAGGGAA 1245 CUCUUCCCUCAGUUUU 1453 GAGdTdT UCG AD-71154 UGGCUGUCCCACCGAA 1246 UUUUUCGGUGGGACAG 1454 AAAdTdT CCC AD-71155 AGGGCCCUGGGGCUGU 1247 GGGACAGCCCCAGGGC 1455 CCCdTdT CCU AD-71156 UCACCCCCGUUAGGGC 1248 AGGGCCCUAACGGGGG 1456 CCUdTdT UGC AD-71157 UCCUGCUCCUGCCGCAC 1249 GGGUGCGGCAGGAGCA 1457 CCdTdT GGA AD-71158 AGUCUCUGUUCCUGCU 1250 AGGAGCAGGAACAGAG 1458 CCUdTdT ACU AD-71159 UGGGGCUUCCAGAGUC 1251 AGAGACUCUGGAAGCC 1459 UCUdTdT CCC AD-71160 UGAGAAAGGUGGGGGG 1252 AAGCCCCCCACCUUUCU 1460 CUUdTdT CG AD-71161 AAUUGAUUCCAGCGAG 1253 UUUCUCGCUGGAAUCA 1461 AAAdTdT AUU AD-71162 UCCUUCUGGGAAAUUG 1254 AAUCAAUUUCCCAGAA 1462 AUUdTdT GGG AD-71163 UGAGCAACUCCCUUCU 1255 CCCAGAAGGGAGUUGC 1463 GGGdTdT UCA AD-71164 UAAAGUCCUGAGUGAG 1256 UUGCUCACUCAGGACU 1464 CAAdTdT UUG AD-71165 UGAAAUGCAUCAAAGU 1257 AGGACUUUGAUGCAUU 1465 CCUdTdT UCC AD-71166 UCUCUGACAGUGUGGA 1258 AUUUCCACACUGUCAG 1466 AAUdTdT AGC AD-71167 UAGGCCAACAGCUCUG 1259 UGUCAGAGCUGUUGGC 1467 ACAdTdT CUC AD-71168 UGGGCCCAGGCGAGGC 1260 UUGGCCUCGCCUGGGC 1468 CAAdTdT CCA AD-71169 UCCCAGAGCCUGGGCC 1261 CUGGGCCCAGGCUCUG 1469 CAGdTdT GGA AD-71170 AGGGCACCCCUUCCCA 1262 CUCUGGGAAGGGGUGC 1470 GAGdTdT CCU AD-71171 UAGCAGGAUCCAGAGG 1263 UGCCCUCUGGAUCCUG 1471 GCAdTdT CUG AD-71172 AAGUGAGGCCACAGCA 1264 UCCUGCUGUGGCCUCA 1472 GGAdTdT CUU AD-71173 AGUUCCCAGGGAAGUG 1265 CCUCACUUCCCUGGGA 1473 AGGdTdT ACU AD-71174 ACACAGGAUGAGUUCC 1266 CUGGGAACUCAUCCUG 1474 CAGdTdT UGU AD-71175 AGCUGCCUCCCCACACA 1267 CCUGUGUGGGGAGGCA 1475 GGdTdT GCU AD-71176 AGCUGUUGGAGCUGCC 1268 GGAGGCAGCUCCAACA 1476 UCCdTdT GCU AD-71177 AGGUCUGGUCAAGCUG 1269 CAACAGCUUGACCAGA 1477 UUGdTdT CCU AD-71178 UGCCCAGGUCUAGGUC 1270 CCAGACCUAGACCUGG 1478 UGGdTdT GCC AD-71179 UGCUGCCCUUUUGGCC 1271 CUGGGCCAAAAGGGCA 1479 CAGdTdT GCC AD-71180 AGCAGCCCCUGGCUGC 1272 AGGGCAGCCAGGGGCU 1480 CCUdTdT GCU AD-71181 ACUGGGUGAUGAGCAG 1273 GGGCUGCUCAUCACCC 1481 CCCdTdT AGU AD-71182 AAAAUGGCCAGGACUG 1274 ACCCAGUCCUGGCCAU 1482 GGUdTdT UUU AD-71183 UCUCAGGCAAGAAAAU 1275 GCCAUUUUCUUGCCUG 1483 GGCdTdT AGG AD-71184 UGGCCUCUUGAGCCUC 1276 CCUGAGGCUCAAGAGG 1484 AGGdTdT CCC AD-71185 AUUGCUCCCUGGGCCU 1277 AAGAGGCCCAGGGAGC 1485 CUUdTdT AAU AD-71186 AGCCCCCUCCCAUUGCU 1278 GGAGCAAUGGGAGGGG 1486 CCdTdT GCU AD-71187 UUCCUCCAUGGAGCCC 1279 AGGGGGCUCCAUGGAG 1487 CCUdTdT GAG AD-71188 AGCUUGGGACACCUCC 1280 GGAGGAGGUGUCCCAA 1488 UCCdTdT GCU AD-71189 UGGUAUUCAAAGCUUG 1281 UCCCAAGCUUUGAAUA 1489 GGAdTdT CCC AD-71190 AAAGGUCUCUGGGGGU 1282 AAUACCCCCAGAGACC 1490 AUUdTdT UUU AD-71191 UGGGAGAGAAAAGGUC 1283 AGAGACCUUUUCUCUC 1491 UCUdTdT CCA AD-71192 UCAGUGAUGGUAUGGG 1284 UCUCCCAUACCAUCAC 1492 AGAdTdT UGA AD-71193 AUCACAAGCCACUCAG 1285 UCACUGAGUGGCUUGU 1493 UGAdTdT GAU AD-71194 UAUCCCAGAAUCACAA 1286 GGCUUGUGAUUCUGGG 1494 GCCdTdT AUG AD-71195 UCUGCGAGGGUCCAUC 1287 UGGGAUGGACCCUCGC 1495 CCAdTdT AGC AD-71196 UCUCUUGCACCUGCUG 1288 UCGCAGCAGGUGCAAG 1496 CGAdTdT AGA AD-71197 UGGGGCUCUGUCUCUU 1289 UGCAAGAGACAGAGCC 1497 GCAdTdT CCC AD-71198 UCAGAGGCUUGGGGGC 1290 AGAGCCCCCAAGCCUC 1498 UCUdTdT UGC AD-71199 UGGGCCCCUUGGGGCA 1291 CUCUGCCCCAAGGGGC 1499 GAGdTdT CCA AD-71200 UCCCCUUUGUGGGCCC 1292 AAGGGGCCCACAAAGG 1500 CUUdTdT GGA AD-71201 UCUGGCCCUUCUCCCCU 1293 AAAGGGGAGAAGGGCC 1501 UUdTdT AGC AD-71202 UAAGAUGUAGGGCUGG 1294 GGGCCAGCCCUACAUC 1502 CCCdTdT UUC AD-71203 UGCUAUGGGAGCUGAA 1295 AUCUUCAGCUCCCAUA 1503 GAUdTdT GCG AD-71204 UUGAGCCAGCGCUAUG 1296 UCCCAUAGCGCUGGCU 1504 GGAdTdT CAG AD-71205 UGGGUUUCUUCCUGAG 1297 UGGCUCAGGAAGAAAC 1505 CCAdTdT CCC AD-71206 UUGAAUGCUGCUUGGG 1298 AACCCCAAGCAGCAUU 1506 GUUdTdT CAG AD-71207 UGGGGUGUGCUGAAUG 1299 CAGCAUUCAGCACACC 1507 CUGdTdT CCA AD-71208 UGGGUUGUCCCUUGGG 1300 CACCCCAAGGGACAAC 1508 GUGdTdT CCC AD-71209 UGUCAUAUGAUGGGGU 1301 ACAACCCCAUCAUAUG 1509 UGUdTdT ACA AD-71210 UGUUGGGCAUGGAGAG 1302 ACCCUCUCCAUGCCCAA 1510 GGUdTdT CC AD-71211 UACAAUCUUAGGUUGG 1303 UGCCCAACCUAAGAUU 1511 GCAdTdT GUG AD-71212 AAAAAACCCACACAAU 1304 AAGAUUGUGUGGGUUU 1512 CUUdTdT UUU AD-71213 AACAUUUUUAAUUAAA 1305 UUUUUUAAUUAAAAAU 1513 AAAdTdT GUU AD-71214 AAAACUUUUAACAUUU 1306 UAAAAAUGUUAAAAGU 1514 UUAdTdT UUU AD-71215 UUUUCAUGUUUAAAAC 1307 AAAGUUUUAAACAUGA 1515 UUUdTdT AAA

TABLE 8 GCK Single Dose Screen in Primary Cynomolgus Hepatocytes DuplexID 20nM_AVG 20nM_STDEV AD-71009 91.7 8.2 AD-71010 85.5 11.8 AD-71011 101.4 19.6 AD-71012 96.3 20.8 AD-71013 102.8 21.7 AD-71014 105.3 47.3 AD-71015 32.6 6.6 AD-71016 98.7 19.1 AD-71017 64.1 16.4 AD-71018 45.9 12.8 AD-71019 39.6 8.5 AD-71020 89.2 23.9 AD-71021 60.4 7.7 AD-71022 100.8 12.7 AD-71023 97.4 33 AD-71024 60.6 18.8 AD-71025 31.1 9 AD-71026 33.6 10.5 AD-71027 46.3 14.6 AD-71028 35.6 9.6 AD-71029 35.6 15.9 AD-71030 30.4 7.9 AD-71031 107.2 29 AD-71032 78.4 15.7 AD-71033 71.8 13.9 AD-71034 39.7 14.3 AD-71035 46.8 11.4 AD-71036 77.6 21.3 AD-71037 37.4 15.2 AD-71038 48.8 19.6 AD-71039 70.1 9.5 AD-71040 65.9 16.2 AD-71041 94.8 18.6 AD-71042 108.4 24.2 AD-71043 35.8 12.2 AD-71044 46.6 10.9 AD-71045 39.4 9.5 AD-71046 35.3 3.9 AD-71047 82 24.4 AD-71048 39.8 10.1 AD-71049 95.3 6.8 AD-71050 151.2 22.4 AD-71051 54 13.5 AD-71052 48.7 8.1 AD-71053 44 10.7 AD-71054 53.4 8.8 AD-71055 39.8 9.4 AD-71056 51.8 34 AD-71057 71 7.1 AD-71058 38.9 3.6 AD-71059 78.1 17.8 AD-71060 54 14.4 AD-71061 108.4 27.2 AD-71062 69.4 6.7 AD-71063 35.1 11 AD-71064 53.1 13.8 AD-71065 94 11.6 AD-71066 149.2 13.3 AD-71067 50.8 15.2 AD-71068 113.4 23.7 AD-71069 44.9 6.4 AD-71070 112.3 24.3 AD-71071 32.7 5.3 AD-71072 40.1 10.2 AD-71073 53 12.5 AD-71074 135.4 25.3 AD-71075 100.8 31.5 AD-71076 35.6 7.3 AD-71077 26.9 4.7 AD-71078 54.4 11.8 AD-71079 48 6.2 AD-71080 96.1 6 AD-71081 105.4 7.9 AD-71082 127 20 AD-71083 117.2 33.2 AD-71084 124.4 20.7 AD-71085 78.5 6.6 AD-71086 30.2 12.7 AD-71087 44.2 2 AD-71088 92.9 21.9 AD-71089 36.1 13.7 AD-71090 40 3.6 AD-71091 58 3.3 AD-71092 62.3 14.9 AD-71093 58.6 18.2 AD-71094 82.7 22.9 AD-71095 116.8 27.6 AD-71096 40.3 11.8 AD-71097 26.6 6.9 AD-69448 34.9 9.3 AD-71098 50.4 7.4 AD-71099 50.7 18.4 AD-71100 23.8 1.7 AD-71101 70.7 18.1 AD-71102 37.2 5.5 AD-71103 98.3 17.6 AD-71104 78.8 22.8 AD-71105 29.6 6.1 AD-71106 26.1 8.1 AD-71107 54.4 9.2 AD-71108 143.2 28.5 AD-71109 116.6 15.9 AD-71110 107.3 21.5 AD-71111 36.8 9 AD-71112 76.4 19.7 AD-71113 71.7 12.9 AD-71114 94.8 22.5 AD-71115 43.3 12.3 AD-71116 58.7 7.9 AD-71117 26.9 7.1 AD-71118 74 15.8 AD-71119 33.8 12.9 AD-71120 30.5 4.3 AD-71121 51.8 7.6 AD-71122 71.8 20.4 AD-71123 45.6 12.4 AD-71124 156.4 6.6 AD-71125 55.4 6.2 AD-71126 102.3 8.5 AD-71127 107.8 18.2 AD-71128 95.6 19 AD-71129 51.6 16.7 AD-71130 28.4 10.3 AD-71131 49 6.5 AD-71132 127.7 25.6 AD-71133 157.5 17.4 AD-71134 38 11.1 AD-71135 62.6 7.6 AD-71136 141.6 20.3 AD-71137 55.9 6.4 AD-71138 37.9 5 AD-71139 125.8 27.6 AD-71140 41.8 2.5 AD-71141 32.8 6.7 AD-71142 40.4 11.5 AD-71143 177.4 27.3 AD-71144 53.5 9.1 AD-71145 41.3 27 AD-71146 105.9 13.9 AD-71147 98.2 27.5 AD-71148 73.6 8.9 AD-71149 159 24.3 AD-71150 157.9 31.1 AD-71151 131.4 4.3 AD-71152 98.2 25.2 AD-71153 66.6 23 AD-71154 134.8 11.9 AD-71155 52.8 4.2 AD-71156 111.4 43.9 AD-71157 37.6 7.7 AD-71158 83.3 16.9 AD-71159 33.7 7.3 AD-71160 44.2 7.2 AD-71161 159.1 28.3 AD-71162 137.2 20.9 AD-71163 23.4 5.4 AD-71164 27.7 2.6 AD-71165 38.1 8 AD-71166 46.4 11.4 AD-71167 53.1 17.6 AD-71168 130.3 14 AD-71169 95.8 24.4 AD-71170 108.8 15.4 AD-71171 57.6 5.3 AD-71172 75 26.8 AD-71173 87.3 24 AD-71174 62.7 24.2 AD-71175 83.6 25.8 AD-71176 26.7 3.5 AD-71177 31 4.7 AD-71178 114.6 20 AD-71179 101.9 25.9 AD-71180 114 12.4 AD-71181 38.2 10.1 AD-71182 32.3 3.2 AD-71183 49.9 7.4 AD-71184 60.6 6.7 AD-71185 30 3 AD-71186 55.7 16.3 AD-71187 72.8 15.2 AD-71188 32.1 2.2 AD-71189 76.4 13.9 AD-71190 34.2 9.1 AD-71191 51.7 9.5 AD-71192 87.5 8.3 AD-71193 110.2 20.9 AD-71194 56.9 12.6 AD-71195 63.1 25.2 AD-71196 38.1 10.3 AD-71197 34 9.8 AD-71198 108.1 12 AD-71199 117.9 11.5 AD-71200 50.1 12.3 AD-71201 38.2 5.8 AD-71202 73.9 3.6 AD-71203 110.3 9.2 AD-71204 140 11 AD-71205 35.8 4.5 AD-71206 28 12.1 AD-71207 22.4 11 AD-71208 54.6 10.9 AD-71209 40.6 14.9 AD-71210 41.5 6.3 AD-71211 54.4 16 AD-71212 122.4 20.7 AD-71213 111.4 13.7 AD-71214 120.1 19.5 AD-71215 98 11.7 

We claim:
 1. A double stranded ribonucleic acid (RNAi) agent for inhibiting expression of a glucokinase (GCK) gene, wherein said double stranded RNAi agent comprises a sense strand and an antisense strand forming a double stranded region, wherein the antisense strand comprises at least 19 contiguous nucleotides differing by no more than 1 nucleotide from the nucleotide sequence of SEQ ID NO:140, wherein substantially all of the nucleotides of said sense strand and substantially all of the nucleotides of said antisense strand are modified nucleotides, and wherein said sense strand is conjugated to a ligand attached at the 3′-terminus.
 2. The double stranded RNAi agent of claim 1, wherein no more than four of the nucleotides of the sense strand are unmodified nucleotides and/or no more than four of the nucleotides of the antisense strand are unmodified nucleotides.
 3. The double stranded RNAi agent of claim 1, wherein at least one of the modified nucleotides is selected from the group consisting of a 3′-terminal deoxy-thymine (dT) nucleotide, a 2′-O-methyl modified nucleotide, a 2′-fluoro modified nucleotide, a 2′-deoxy-modified nucleotide, a locked nucleotide, an unlocked nucleotide, a conformationally restricted nucleotide, a constrained ethyl nucleotide, an abasic nucleotide, a 2′-amino-modified nucleotide, a 2′-O-allyl-modified nucleotide, 2′-C-alkyl-modified nucleotide, 2′-hydroxyl-modified nucleotide, a 2′-methoxyethyl modified nucleotide, a 2′-O-alkyl-modified nucleotide, a morpholino nucleotide, a phosphoramidate, a non-natural base comprising nucleotide, a tetrahydropyran modified nucleotide, a 1,5-anhydrohexitol modified nucleotide, a cyclohexenyl modified nucleotide, a nucleotide comprising a 5′-phosphorothioate group, a nucleotide comprising a 5′-methylphosphonate group, a nucleotide comprising a 5′ phosphate or 5′ phosphate mimic, a nucleotide comprising vinyl phosphate, a nucleotide comprising adenosine-glycol nucleic acid (GNA), a nucleotide comprising thymidine-glycol nucleic acid (GNA) S-Isomer, a nucleotide comprising 2-hydroxymethyl-tetrahydrofurane-5-phosphate, a nucleotide comprising 2′-deoxythymidine-3′phosphate, a nucleotide comprising 2′-deoxyguanosine-3′-phosphate, and a terminal nucleotide linked to a cholesteryl derivative or a dodecanoic acid bisdecylamide group.
 4. The double stranded RNAi agent of claim 1, further comprising at least one phosphorothioate internucleotide linkage.
 5. The double stranded RNAi agent of claim 1, wherein each strand is no more than 30 nucleotides in length.
 6. The double stranded RNAi agent of claim 1, wherein at least one strand comprises a 3′ overhang of at least 1 nucleotide.
 7. The double stranded RNAi agent of claim 1, wherein the ligand is an N-acetylgalactosamine (GalNAc) derivative.
 8. The double stranded RNAi agent of claim 7, wherein the ligand is


9. A cell containing the double stranded RNAi agent of claim
 1. 10. A pharmaceutical composition for inhibiting expression of a glucokinase (GCK) gene comprising the double stranded RNAi agent of claim
 1. 11. A method of inhibiting expression of a glucokinase (GCK) gene in a cell, the method comprising: (a) contacting the cell with the double stranded RNAi agent of claim 1 or the pharmaceutical composition of claim 10; and (b) maintaining the cell produced in step (a) for a time sufficient to obtain degradation of the mRNA transcript of a GCK gene, thereby inhibiting expression of the GCK gene in the cell.
 12. The method of claim 11, wherein said cell is within a subject.
 13. The method of claim 12, wherein the subject is a human.
 14. The method of claim 13, wherein the human subject suffers from a disease or disorder that would benefit from reduction in GCK expression.
 15. The method of claim 14, wherein the disease or disorder is a glycogen storage disease (GSD).
 16. A method of treating a subject having a disease or disorder that would benefit from reduction in expression of a glucokinase (GCK) gene, comprising administering to the subject a therapeutically effective amount of the double stranded RNAi agent of claim 1 or the pharmaceutical composition of claim 10, thereby treating said subject.
 17. The method of claim 16, wherein the disease or disorder is a glycogen storage disease (GSD).
 18. The method of claim 16, wherein the administration of the double stranded RNAi agent to the subject causes an increase in blood glucose levels, a decrease in plasma lactate levels, a decrease in plasma uric acid levels, a decrease in plasma triglyceride levels, a decrease in total plasma cholesterol levels, and/or a decrease in GCK protein accumulation.
 19. The method of claim 16, further comprising administering a sodium-glucose co-transporter 2 (SGLT2) inhibitor to the subject.
 20. A method of inhibiting the expression of a glucokinase (GCK) gene in a subject, the method comprising administering to said subject a therapeutically effective amount of the double stranded RNAi agent of claim 1 or the pharmaceutical composition of claim 10, thereby inhibiting the expression of GCK in said subject.
 21. The double stranded RNAi agent of claim 1, wherein each of the sense strand and the antisense strand independently is 21 to 23 nucleotides in length.
 22. The double stranded RNAi agent of claim 1, wherein the sense strand is 21 nucleotides in length and the antisense strand is 23 nucleotides in length.
 23. The double stranded RNAi agent of claim 1, wherein the iRNA agent targets nucleotides 563-587 of SEQ ID NO:1.
 24. The double stranded RNAi agent of claim 1, wherein the antisense strand comprises at least 20 contiguous nucleotides differing by no more than 3 nucleotides from the nucleotide sequence of 5′-UUGAUGCUUGUCCAGGAAGUCGG-3′ (SEQ ID NO: 140).
 25. The double stranded RNAi agent of claim 1, wherein the antisense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from the nucleotide sequence of 5′-AUCUGAUGCUUGUCCAGGAAGUC-3′ (SEQ ID NO: 141).
 26. The double stranded RNAi agent of claim 1, wherein the sense strand comprises the nucleotide sequence 5′-GACUUCCUGGACAAGCAUCAA-3′ (SEQ ID NO: 51) and the antisense strand comprises the nucleotide sequence 5′-UUGAUGCUUGUCCAGGAAGUCGG-3′ (SEQ ID NO: 140).
 27. The double stranded RNAi agent of claim 1, wherein the sense strand comprises the nucleotide sequence of 5′-CUUCCUGGACAAGCAUCAGAU-3′ (SEQ ID NO: 52) and the antisense strand comprises the nucleotide sequence 5′-AUCUGAUGCUUGUCCAGGAAGUC-3′ (SEQ ID NO: 141).
 28. The double stranded RNAi agent of claim 26, wherein the sense strand comprises the nucleotide sequence 5′-gsascuucCfuGfGfAfcaagcaucaa 3′ (SEQ ID NO:229) and the antisense strand comprises the nucleotide sequence 5′-usUfsgauGfcUfUfguccAfgGfaagucsgsg 3′ (SEQ IDNO:318), wherein a, g, c, and u are 2′-O-methyl (2′-OMe) modified A, G, C and U nucleotides, respectively; Af, Gf, Cf and Uf are 2′ fluoro A, G, C and U modified nucleotides, respectively; and s is a phosphorothioate linkage.
 29. The double stranded RNAi agent of claim 27, wherein the sense strand comprises the nucleotide sequence 5′-csusuccuGfgAfCfAfagcaucagau-3′ (SEQ ID NO:230) and the antisense strand comprises the nucleotide sequence 5′-asUfscugAfuGfCfuuguCfcAfggaagsusc-3′ (SEQ IDNO:319), wherein a, g, c, and u are 2′-O-methyl (2′-OMe) modified A, G, C and U nucleotides, respectively; Af, Gf, Cf and Uf are 2′ fluoro A, G, C and U modified nucleotides, respectively; and s is a phosphorothioate linkage.
 30. The double stranded RNAi agent of claim 28 or 29, further comprising a ligand.
 31. The double stranded RNAi agent of claim 30, wherein the ligand is


32. The double stranded RNAi agent of claim 8 or 31, wherein the double stranded RNAi agent is conjugated to the ligand as shown in the following schematic

and, wherein X is O or S.
 33. The double stranded RNAi agent of claim 32, wherein the X is O.
 34. The double stranded RNAi agent of claim 2, wherein all of the nucleotides of the sense strand are modified nucleotides and/or all of the nucleotides of the antisense strand are modified nucleotides.
 35. The double stranded RNAi agent of claim 1, wherein each strand is independently 19-30 nucleotides in length.
 36. The double stranded RNAi agent of claim 1, wherein each strand is independently 19-25 nucleotides in length.
 37. The double stranded RNAi agent of claim 1, wherein at least one strand comprises a 3′ overhang of at least 2 nucleotides.
 38. The double stranded RNAi agent of claim 1, wherein at least one of said modified nucleotides is selected from the group consisting of a 2′-O-methyl modified nucleotide and a 2′fluoro modified nucleotide. 